Methods and compositions for treating cancer

ABSTRACT

Methods for treating cancer are disclosed which comprise administering to a subject T cells which have been pretreated ex vivo or in vitro with a fatty acid catabolism promoter to condition the T cell to use fatty acids rather than glucose for energy production. Still other methods comprise co-administering to a subject having a cancer characterized by a solid tumor (a) an immunotherapeutic composition targeting an antigen or ligand on the tumor cell; and (b) a compound or reagent that promotes the use of fatty acid catabolism by tumor antigen-specific T cells in the tumor microenvironment and/or T cells pretreated ex vivo with the fatty acid catabolism promoter to condition the T cell to use fatty acids rather than glucose for energy production for adoptive cell transfer. Both methods may also employ co-administration of a checkpoint inhibitor.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled“WST161PCT_ST25.txt” and dated Jan. 9, 2017.

BACKGROUND OF THE INVENTION

Despite recent progress in cancer immunotherapy,^(7, 29) cures remainrare even for 14highly immunogenic tumors such as melanomas. The tumormicroenvironment (TME) poses significant metabolic challenges to TILsdue to disorganized vascularization, presence of toxic products derivedfrom tumor and stromal cells and lack of nutrients and oxygen (O₂).² Tworecent papers show that lack of glucose impairs effect functions oftumor infiltrating T lymphocytes (TILs).^(6,14) Tumors not only lackglucose but also oxygen. Functional declines of tumor-infiltrating Tlymphocytes (TILs) dampen the efficacy of immunotherapy for solidtumors. This is thought to reflect in part their exhaustion driven bycontinuous antigenic stimulation. Adoptive transfer of ex vivo expandedTILs may affect regression of large melanomas³⁸. Nevertheless,traditional vaccines that aim to induce such T cells have largely beenineffective.⁹ Exhaustion of tumor antigen (TA)-specific CD8+Tcells^(1,3) is characterized by their enhanced expression ofco-inhibitors, decreased levels of the transcription factor T-bet andloss of effector functions³⁶ following chronic tumor-derived antigenstimulation.²² T cell exhaustion has been implicated to cause failuresof active immunotherapy for solid tumors.

TILs require energy to eliminate tumor cells. Upon activation T cellsenhance energy production through glycolysis,³⁴. Glycolysis is lessefficient than oxidative phosphorylation (OXPHOS), but provides buildingblocks for biomass formation and cell proliferation. Tumor cells alsouse glycolysis,¹³ which may lead to glucose (Glu) depletion within theTME.^(6,14) T cells with limited access to Glu have to rely on OXPHOS toproduce energy. Although many substances including fatty acids (FAs) canfuel OXPHOS, it requires O₂, which can become limiting within tumors dueto insufficient blood supply.¹⁹ TILs therefore face dual metabolicjeopardy, which drives their functional exhaustion and thereby impairsthe efficacy of cancer immunotherapy.

Thus, metabolism plays an important role in modulating T cell effectorfunctions. How TILs adapt to the metabolic constraints within the TME,including glucose and oxygen deprivation, and how these constraintsaffect TIL ability to combat tumor progression remains poorlyunderstood.

There remains a need in the art for new and effective tools and methodsto facilitate treatment and prophylactic therapies for cancer.

SUMMARY OF THE INVENTION

In one aspect, a method for treating cancer comprises administering to asubject having a cancer characterized by a solid tumor a compound orreagent that promotes the use of fatty acid catabolism by tumorantigen-specific T cells in the tumor microenvironment (referred tovariously as “a fatty acid catabolism promoter” or “fatty acidcatabolism-promoting compound”). In another embodiment, this methodinvolves co-administering a checkpoint inhibitor in the form of anantibody or a small molecule.

In another aspect, a method for treating cancer comprises administeringto a subject having a cancer a T cell that is pretreated or conditionedex vivo or in vitro with a compound or reagent that promotes the use offatty acid catabolism rather than glucose for energy production by thepre-treated T cells.

In another aspect, a method for treating cancer comprisesco-administering to a subject having a cancer characterized by a solidtumor an immunotherapeutic composition targeting an antigen or ligand onthe tumor cell; and a compound or reagent that promotes the use of fattyacid catabolism by tumor antigen-specific T cells in the tumormicroenvironment. In another embodiment, this method involvesco-administering a checkpoint inhibitor in the form of an antibody or asmall molecule.

In another aspect, a method for treating cancer comprisesco-administering to a subject having a cancer characterized by a solidtumor an immunotherapeutic composition targeting an antigen or ligand onthe tumor cell; and selected T cells pretreated ex vivo or treated upontransfer with a compound or reagent that promotes the use of fatty acidcatabolism by tumor antigen-specific T cells to condition the T cell touse fatty acids rather than glucose for energy production prior to orupon adoptive cell transfer. In another embodiment, this method involvesco-administering a checkpoint inhibitor in the form of an antibody or asmall molecule.

In another aspect, a method for treating cancer comprisesco-administering the immunotherapeutic composition, the fatty acidcatabolism promoter and the selected T cells identified herein. Inanother embodiment, this method involves co-administering a checkpointinhibitor in the form of an antibody or a small molecule.

In another aspect, a method for treating cancer comprises administeringto a subject having a cancer a T cell that is pretreated ex vivo or invitro with a compound or reagent that promotes the use of fatty acidcatabolism by the T cells. The fatty acid catabolism promoting compoundor reagent conditions the T cell to use fatty acids rather than glucosefor energy production. This pretreatment and conditioning enhances theimmune function of the T cell(s) once the T cell(s) are readministered,i.e., by adoptive therapy, to the subject. In another embodiment, thismethod involves co-administering a checkpoint inhibitor in the form ofan antibody or a small molecule in combination with, or sequentiallywith, administration of the pre-treated, conditioned T cell. In anotheraspect, a method of modifying a T cell comprises pretreating the T cellex vivo or in vitro with a compound or reagent that conditions the cellto use fatty acid catabolism for energy production by the T cells.

In another aspect, a method of enhancing the survival of a T cell, e.g.,an autologous T cell, a chimeric antigen receptor-T cell, a chimericendocrine receptor-T cell or ex vivo expanded tumor antigen-specific Tcells comprising treating the T cell(s) ex vivo with a compound orreagent that promotes the use of fatty acid catabolism for energyproduction by tumor antigen-specific T cells in the tumormicroenvironment before or upon adoptive cell transfer to a subjecthaving a solid tumor.

In yet another aspect, a therapeutic regimen is provided for thetreatment of cancer comprising administering to a subject having acancer characterized by a solid tumor a single dose of animmunotherapeutic composition targeting an antigen or ligand on thetumor cell on a day 1 of treatment. In this regimen, the subject isthereafter administered a compound or reagent that promotes the use offatty acid catabolism by tumor antigen-specific T cells in the tumormicroenvironment. The first dose of the fatty acid catabolism-promotingcompound of reagent begins on any of day 0, 1, 2, 3, 4 or 5 oftreatment. Also involved in this regimen is the step of administeringthe fatty acid catabolism-promoting compound or reagent daily from thebeginning day of treatment of immunotherapeutic composition until a dayoccurring between day 7 to day 30 of treatment.

In still a further aspect, a composition is provided for adoptivetransfer to a mammalian subject comprising a T cell that has beenpretreated ex vivo or in vitro with a compound or reagent thatconditions the cell to use fatty acid catabolism for energy productionby the T cells.

Other aspects and advantages of these compositions and methods aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing tumor growth in mice that received AdC68-gD(Control, Co) or AdC68-gDMelapoly mixed with AdC68-gDE7 (Vaccine) 3 daysafter tumor challenge (n=6-18/group). CD8+TILs become functionallyimpaired within the tumor microenvironment (TME).

FIG. 1B are a bar graph (left) showing the ratio of antigen-specificCD8+T cell frequencies from spleens and tumors producing factorsharvested 1 mo over those harvested 2 wk after tumor challenge; and abar graph (right) showing absolute frequencies of specific CD8+ T cellsat 1 mo after challenge from spleens or tumors producing 3 or 2 factors.Production of IFN-γ, granzyme B (GrzmB) and perforin were measured.n=5-7/group.

FIG. 1C are 4 graphs showing MFI (mean-SEM) of CD62L, CD127, KLRG1 andEomes (left to right) on/in specific CD8+ T cells from spleens.n=5-7/group. For all figures, results are shown as mean values withstandard errors of mean (SEM). (*) Indicates significant differencesbetween groups; *p≤0.05-0.01, ** p≤0.01-0.001, *** p≤0.001-0.0001, ****p≤0.0001.

FIG. 1D shows 3 bar graphs of characteristics of human T cells invarious cancers on expression of PD-1, PPAR-α, and Bodipy C16 forcomparison with the studies herein.

FIGS. 2A-2C illustrate that CD8+TILs increasingly experience metabolicstress within the TME.

FIG. 2A illustrates MFI values (mean-SEM) of mitochondrial membranepotential (MMP) and mitrochondrial reactive oxygen species (MROS) stainsin Trp-1- and E7-specific CD8+T cells from spleens and tumors harvested2 weeks or 1 month after challenge (n=5 mice/group, representative of 3experiments). MMP allows for formation of proton gradients used for ATPsynthesis by OXPHOS. MROS is a superoxide highly toxic byproduct ofOXPHOS mainly generated by complex I and III of the mitochondrialelectron transport chain.

FIG. 2B shows a bar graph reporting results of quadrant gating offrequencies of Trp-1- and E7-specific CD8+T cells with high or lowlevels of MMP and MROS from mice bearing 1-mo tumors or CD44− T cells.(−) not significant or (*) significant shown above the bars in the graphare arranged so that the 1^(st) to 4^(th) bars show differences betweenMMP^(hi)MROS^(lo) cells, MMP^(hi)MROS^(hi) cells, MMP^(lo)MROS^(hi)cells and MMP^(lo)MROS^(lo) cells, respectively.

FIG. 2C illustrates MFI (mean-SEM) of HIF-1α and Glut1 expression in/onspecific Trp-1- and E7-specific CD8+ T cells from spleens and tumors ofmice bearing 1-mo tumors (n=5/group, representative of 2 assays). Allbar graphs show mean-SEM. Representative histograms for samples fromspleen and tumor were compared (data not shown).

FIGS. 3A-3D illustrate the effect of hypoxia on characteristics ofactivated CD8+T cells. HIF-1α knock down reduces LAG-3 expression andimproves melanoma associated antigen (MAA)-specific CD8⁺T cellfunctions.

FIG. 3A shows in the left bar graph the MFI (mean-SEM) values of HIF-1αexpression in/on Trp-1-specific CD8⁺TILs transduced with control (coRNA) or HIF-1α shRNA (shRNA) vector. The right two bar graphs show theMFI (mean-SEM) values of co-inhibitors PD-1 (left) and LAG-3 (right)expression by Trp-1-specific CD8⁺TILs transduced with control (co RNA)or HIF-1α shRNA (shRNA) vector. Representative histograms for samplestransduced with control or HIF-1α shRNA vector were compared (data notshown). HIF-1α correlates with co-inhibitor LAG-3 expression in vitroand in vivo.

FIG. 3B illustrates % of MAA-specific CD8+TILs transduced with control(co RNA) or HIF-1α shRNA (shRNA) expressing vectors producing individualfactors, GzmB, IFN-γ or Perforin (from left to right). All bar graphsshow mean-SEM.

FIG. 3C is a bar graph illustrating % of lentivector transduced cellsproducing 3, 2 or 1 (from top to bottom of each bar) of the factors(n=5-7 mice/group). * within ( ) indicates significant difference in sumof responses, * out of ( ) left to right: significant difference in 3,2, 1 functions. All bar graphs show mean-SEM. HIF-1α knock-downincreases CD8+ TIL functions.

FIGS. 4A-4I show that limited access to Glu and oxygen forces activatedCD8+TILs to enhance FA catabolism. In late stage melanoma, glucose andoxygen become limiting.

FIG. 4A is a bar graph showing Glu concentrations in plasma andinterstitial fluid of tumors (n=3/group). Data are shown as mean-SEM.

FIG. 4B shows relative transcript levels in CD8+T cells stimulated inGal or Glu+2-DG medium in comparison to those of CD8⁺T cells cultured inGlu medium under hypoxia in the upper two rows (n=3-4 samples/group). Inthe middle 2 rows, relative transcript levels are shown for Trp-1- andE7-specific CD8+TILs from 1-mo-old tumors compared to CD8⁺TILs of thecorresponding antigen specificity from 2 week-old tumors (n=4-5samples/group). Lower 2 rows show relative transcript levels for Trp-1-and E7-specific CD8+T splenocytes harvested 3 mo vs. 2 wk aftervaccination. Line code shown to the right of the map compares changes intranscript levels between in vitro and in vivo samples. Transcriptsinvolved in lipid metabolism are enhanced in metabolically stressed Tcells in vitro and in vivo.

FIG. 4C is a bar graph showing the intensity of glycolysis metabolitesin CD44⁺CD8⁺TILs from 1 month-old tumors compared to those ofCD44⁺CD8⁺TILs from 2-week old tumors. Experiments were conducted twicewith 2-3 pooled samples collected from ˜30 mice/sample/experiment. Dataare shown as mean values.

FIG. 4D shows ¹³C₆-Glu contribution to TCA cycle metabolites inCD44⁺CD8⁺TILs from mice bearing 1-mo tumors normalized to those from2-wk tumors (left) or spleens of the same mice harvested at 1 mo afterchallenge (right). Experiments were conducted twice with 2-3 pooledsamples collected from ˜30 mice/sample/experiment. Data are shown asmean values.

FIG. 4E shows the intensity of FA metabolites in CD44⁺CD8⁺TILs from 1month-old tumors compared to those of CD44⁺CD8⁺TILs from 2-week tumors.Experiments were conducted twice with 2-3 pooled samples collected from˜30 mice/sample/experiment. Data are shown as mean values.

FIG. 4F are three bar graphs showing normalized ¹³C₁₆-palmitatecontribution to TCA cycle metabolites in CD44⁺CD8⁺ T cells from 1-mo vs.2-wk tumors (left) or 1-mo tumors vs. 1-mo spleens (middle) or spleensat 1 mo vs. 2 wk after tumor challenge (right). Experiments wereconducted twice with 2-3 pooled samples collected from ˜30mice/sample/experiment. Data are shown as mean values.

FIG. 4G is a bar graph showing relative intensity of free FA species inthe tumor interstitial fluid shown as ratio of results obtained from1-mo over 2-wk tumors (n=2-3 samples/group). Data are shown as mean-SEM.

FIG. 4H is a graph showing uptake of a boron-dipyrromethene fluorescentdye (Bodipy) C₁₆ by freshly isolated Trp-1- or E7-specific CD8⁺T cellsfrom spleens or tumors harvested 2 weeks or 1 month after tumorchallenge tested directly ex vivo n=5/group. Representative histogramsfor samples from spleen and tumor harvested 1 month after tumorchallenge were compared (data not shown). Lines with * above barsindicate significant differences. Data are shown as mean-SEM.

FIG. 4I shows a bar graph represented MFI for Cpt1a in Trp-1- andE7-specific CD8⁺T cells from spleens or tumors harvested 2 weeks or 1month after tumor challenge. Representative histogram are not shown.n=5/group. Lines with * above bars indicate significant differences.Data are shown as mean-SEM. FA catabolism is enhanced in CD8+ TILsduring tumor progression.

FIGS. 5A to 5F show that metabolism and effector functions of CD8+TILsare independent of PD-1. Tumor-bearing mice vaccinated on day 3, weretreated with isotype or anti-PD-1 starting on day 10 after vaccination.Vaccinated tumor bearing mice were treated with isotype control (Iso) oranti-PD-1 antibody (α-PD-1) every 3rd day.

FIG. 5A shows the MFI of PD-1 and pAkt on/in specific CD8+TILs at 1 moafter tumor challenge. n=5-7 mice/group, Data is shown as mean-SEM.

FIG. 5B shows MFI of markers (CD62L, CD127 and Eomes, panels from leftto right) on/in specific CD8+TILs from 1-mo tumors. n=5-7 mice/group.Data is shown as mean-SEM.

FIG. 5C shows a ¹³C₁₆-palmitate contribution to TCA cycle metabolites inCD44⁺CD8⁺T cells from α-PD-1 treated (dark grey) normalized toiso-treated 1-mo tumors (light grey). n=2-3 samples/group. Each sampleis pooled from 20-30 mice. Data is shown as mean values.

FIG. 5D shows the intensity of ketone bodies in CD44⁺CD8⁺T cells fromα-PD-1 treated (dark grey) compared to iso-treated 1-mo tumors (lightgrey). n=2-3 samples/group. Each sample is pooled from 20-30 mice. Datais shown as mean values.

FIG. 5E shows ¹³C₆-Glu contribution to TCA cycle metabolites inCD44⁺CD8⁺T cells from α-PD-1 treated (dark grey) normalized toiso-treated 1-mo tumors (light grey). n=2-3 samples/group. Each sampleis pooled from 20-30. Data is shown as mean values. These results showthat PD-1 blockade does not affect CD8+ TILs' metabolism.

FIG. 5F shows frequencies of specific CD8⁺TILs from 1-mo tumors of micetreated with iso or α-PD-1 producing 3, 2 or 1 factors (from top tobottom of each bar). n=11-15 mice/group. Data is shown as mean-SEM.

FIGS. 6A-6J illustrate that promoting FA catabolism improves CD8+TILfunctions without reducing PD-1 expression.

FIG. 6A is a cartoon of an experimental setup of the in vivo studyshowing drugs that target different pathways of FA catabolism.

FIG. 6B are bar graphs showing MFI of markers (PD-1, T-bet, CD62L,Cd127, KLRG1 and total FoxO1, panels from left to right) on/in donorCD8+T cells from mice treated with diluent (Dil.) or FF before transfer.n=8-10/group. Data is shown as mean-SEM.

FIG. 6C is a bar graph showing functions of CD8+T cells from spleens ofdonor mice treated with Dil. or FF before transfer as % of cellsproducing 3, 2 and 1 factors (from top to bottom of each bar).n=8-10/group. Data is shown as mean-SEM.

FIG. 6D is a bar graph showing basal OCR of CD8+T cells from spleens ofdonor mice fed with Dil. or FF. Some samples were incubated withEtomoxir (Eto) (n=5-6 mice/group). Data is shown as mean-SEM.

FIG. 6E is a bar graph showing MFI of PD-1 on Dil.- or FF-treated donorCD8+TILs (n=6/group). Data is shown as mean-SEM.

FIG. 6F is a bar graph showing % specific CD8+TILs from Dil.- or FFtreated donors producing 3, 2, or 1 factors (from top to bottom of eachbar). (−) or (*) on top of each bar indicates significant differences insum of the responses. * from bottom to top: differences in producing 1,2 or 3 factors. Data is shown as mean-SEM.

FIG. 6G is a graph showing the tumor weight 2 weeks after cell transfer(n=5/group). Data is shown as mean±SEM.

FIG. 6H is a cartoon of an experimental design of PD-1 blockade combinedwith transfer of FF- or Dil.-treated T cells.

FIG. 6I is a graph showing tumor progression indicated as tumor volumein mice that received either FF- or Dil.-treated cells and either iso orα-PD-1 treatment after cell transfer. n=6-7/group. Data is shown asmean±SEM.

FIG. 6J is a set of bar graphs showing PD-1 expression on donor-derivedFF- or Dil.-treated CD8+TILs recovered from recipients treated with isoor α-PD-1. n=6-7/group. Data is shown as mean-SEM.

FIGS. 7A-7C illustrate that reducing FA catabolism decreases PD-1expression and impairs CD8⁺TILs.

FIG. 7A is a cartoon showing experimental setup for the in vivo study.

FIG. 7B is MFI of PD-1 expression on wt and PPAR-α KO donor Trp-1- andE7-specific CD8⁺TILs (n=6/group, representative of 2 assays). Data isshown as mean-SEM. Decreasing FA catabolism reduces PD-1 expression.

FIG. 7C shows the % of Trp-1- and E7-specific CD8⁺TILs from the twogroups of donor mice producing 3, 2, and 1 factors (from top to bottomof each bar). * outside of ( ) indicates differences producing 1-3functions (bottom to top). * within ( ) indicates differences in overallfrequencies. Flow blots illustrating functions of donor-derived Trp-1-and E7-specific CD8⁺TILs from each group are not shown. n=6/group. Datais shown as mean-SEM. Decreasing FA catabolism dampens CD8+ TILfunctions.

FIGS. 8A-8H show that hypoxia affects CD8+T cell metabolism,differentiation and functions.

FIG. 8A illustrates lymphoblast formation; normalized change of % ofactivated live CD8+T cells forming blasts by day 4 of culture underhypoxia (H, lightgrey) compared to those cultured under normoxia (N,dark grey). * on top of each bar indicates significant differencescompared to values obtained from samples cultured in Glu medium undernormoxia. (n=4-5 samples/group, representative of 3-5 experiments). Datais shown as mean-SEM.

FIG. 8B shows normalized change of MFI values for HIF-1α and Glut1expression (stains) in/on activated CD8+T cells cultured under normoxia,N or hypoxia, H (n=4-5 samples/group, representative of 3-5experiments). * on top of each bar indicates significant differencescompared to values obtained from samples cultured in Glu medium undernormoxia. Data is shown as mean-SEM.

FIG. 8C aregraphs showing basal OCR (left) and ECAR (right) rate of day4 activated CD8+T cells cultured under normoxia (N) or hypoxia (H).(n=4-5/condition, representative of 3-5 experiments). Data is shown asmean-SEM.

FIG. 8D shows normalized change of MFI of PD-1, LAG-3 and T-bet (fromleft to right) in/on activated cells (n=4-5/condition, representative of3-5 experiments). Data is shown as mean-SEM.

FIG. 8E shows production of individual functions of activated cellscultured under hypoxia (H) with data normalized to normoxia (N).Functions of CD8+T cells shown as change of % of CD8⁺CD44⁺T cellsproducing IFN-γ, GzmB and perforin factors (their representative flowplots are not shown). (n=4-5/condition, representative of 3-5experiments). Data is shown as mean-SEM.

FIG. 8F shows blast formation; normalized % change of live IL-2maintained resting CD8+T cells forming blasts under hypoxia (H, white)compared to those cultured under normoxia (H, gray) (n=3-4samples/group, representative of 2 experiments). Data is shown asmean-SEM.

FIG. 8G shows normalized expression change of PD-1, LAG-3 and T-beton/in IL-2 maintained resting cells subjected to hypoxia (H, white)compared to those cultured under normoxia (N, grey). n=6/condition,representative of 2 experiments. * above bars indicate significantdifferences between samples kept under normoxia and those subjected tohypoxia. Data is shown as mean-SEM.

FIG. 8H shows production of individual factors by IL-2 maintainedresting CD8+T cells cultured under hypoxia (H, white) compared to thosecultured under normoxia (N, grey). % change of IL-2 maintained restingCD8+T cells producing IFN-γ, GzmB and perforin were quantified. Data isshown as mean-SEM.

FIG. 9A-9E show the effects of glucose limitation on metabolism,differentiation and effector functions of CD8+T cells activated in vitrounder normoxia (N) or short-term hypoxia (H). Indicated (in Y-axistitle) values are normalized to those obtained with Glu cultures kept atnormoxia, which are set at 100 (dotted black lines). The stippled lightgray lines show results for cells cultured in Glu and subjected tohypoxia normalized to results for cells cultured in Glu and undernormoxia. Galactose/2-Deoxy-D glucose (2-DG) mimics lack of glucose. Alldata are shown as mean-SEM.

FIG. 9A shows normalized basal OCR change (right panel) andextracellular acidification rate (ECAR) change (left panel) for CD8+Tcells stimulated in regular Glu-rich medium, Glu with 2-DG, or mediumsupplemented with Gal instead of Glu for 4 days. (n=4 samples/group,pooled from 20-30 mice/experiment and representative of 3 experiments).

FIG. 9B shows the OCR to ECAR ratios at baseline for CD8+T cellsstimulated in regular Glu-rich medium, Glu with 2-DG, or mediumsupplemented with Gal instead of Glu for 4 days. (n=4 samples/group,pooled from 20-30 mice/experiment and representative of 3 experiments).Lack of glucose increases the OCR/ECAR ratio.

FIG. 9C show normalized changes of MFI values for PD-1 expression onCD8+T cells activated under normoxia (N, dark grey) or hypoxia (H, lightgrey) (n=4-5/condition, representative of >5 assays). Lack of glucoseincreases PD-1 expression. Representative histograms are not shown.

FIG. 9D show normalized changes of MFI values of T-bet (n=4-5,representative of >3 assays).

FIG. 9E shows Left: % of cells producing 3, 2 and 1 factors (from top tobottom of each bar) over all CD44+CD8+ T cells cultured under differentconditions (N, normoxia; H, hypoxia). Right: Same data as leftillustrating differences in % of cells producing 3, 2 and 1 factors(from bottom to top for the left two bars and from top to bottom for theright two bars) in Glu+2-DG or Gal medium compared to those of cells inGlu medium with the corresponding O₂ supply. Statistics on each barindicates difference in % of cells producing 3, 2 and 1 function (bottomto top). Representative flow plots for IFN-γ and GzmB production are notshown. Lack of glucose reduces CD8+ T cell functions under normoxia andless pronounced under hypoxia.

FIGS. 10A-10E show the effects of glucose and O₂ limitation on FAcatabolism of CD8+T cells activated in vitro. All data are shown asmean-SEM.

FIG. 10A shows the relative intensity of fatty acid catabolism-relatedmetabolites in CD8+T cells stimulated for 4 days in vitro underdifferent conditions. Normoxia (N, dark gray); Hypoxia (H, light grey);compared to those of cells cultured under Glu, N (enriched CD8+T cellswere pooled from 20-30 mice for each experiment, representative of 2assays).

FIG. 10B shows normalized contribution change of ¹³C₆-Glu/Gal-derived¹³C carbon to metabolites of the TCA cycle in cells cultured indifferent media under different conditions (Normoxia, N, dark gray;Hypoxia, H, light grey) compared to those cultured in Glu medium undernormoxia, N (set as 100, indicated by dotted line). Data are shown asrelative mean % change of labeling (enriched CD8+T cells were pooledfrom 20-30 mice for each experiment, representative of 2 assays). (*) ontop of each bar indicates significant differences compared to cellscultured under Glu, N.

FIG. 10C shows normalized contribution change of ¹³C₆-palmitate-derived¹³C-carbon to metabolites of the TCA cycle in cells cultured indifferent media under different conditions (Normoxia, N, dark gray;Hypoxia, H, light grey) comparing to those cultured under Glu, N (set as100, indicated by dotted line). Data are shown as relative mean % changeof labeling (enriched CD8+T cells were pooled from 20-30 mice for eachexperiment, representative of 2 assays). (*) on top of each barindicates significant differences compared to cells cultured under Glu,N. More FA-derived carbon contributes to TCA metabolites and amino acidsindicating enhanced use of FA for ATP and biomass production underhypoglycemia and hypoxia.

FIG. 10D illustrates the uptake of Bodipy FL C₁₆ (fluoresent free FA) bycells cultured in Glu (dark gray) or Gal (light gray) media undernormoxia (N) or hypoxia (H). Histograms of representative samplessubjected to hypoxia are not shown. (n=5/group, representative of twoexperiments).

FIG. 10E shows basal OCR of FAO due to consumption of exogenous (darkgray bars) and endogenous (light gray bars) FAs by CD8+ T cellsstimulated in vitro in Glu or Gal media (n=3 samples, pooled from 15mice/group). FA catabolism increases when glucose is limiting.

FIGS. 11A-11D show that anti-PD-1 treatment affects tumor cellmetabolism, i.e., increasing Glu concentration in the tumor interstitialfluid and the tumor cells' Glu metabolism.

FIG. 11A is a bar graph showing that Glu concentration in the tumorinterstitial fluid from the indicated mice that had received the isotypecontrol (Iso, white) or the anti PD-1 antibody (α-PD-1, grey). Data isshown as mean-SEM.

FIG. 11B is a schematic that illustrates the ¹³C₆-glucose metabolism oftumor cells using catabolic pathway by contributing two ¹³C carbons tocitrate and TCA cycle intermediate, α-ketoglutarate, or using anabolicpathway by contributing three ¹³C carbons to oxaloacetate and citrateand the purine synthesis pathway intermediate AICAR.

FIG. 11C illustrates three bar graphs showing the results for ¹³C₆-Glutracing of cells isolated from day 20 tumors of NSG mice. Incorporationof 2 and 3 carbons are shown indicating the use of Glu for catabolic oranabolic downstream reactions. Data is shown as mean-SEM.

FIG. 11D is a bar graph reporting the MFI of PD-1 (grey) and PD-L1(black) expression on the identified tumor cells. Ligation of PD-1 toPD-L1 on tumor cells increases their resistance to apoptosis or T cellmediated cytolysis.

FIGS. 12A-12D illustrate that manipulating FA metabolism of activatedCD8+T cells in vitro affects their differentiation and functions.

FIG. 12A shows Bodipy C₁₆ uptake by CD8+T cells stimulated in vitro inGlu or Gal and subjected to short-term hypoxia (H) with the addition ofFF (FF, light grey) compared to those of cells cultured under samecondition with the addition of diluent (Co, dark grey). * above barsindicate significant differences between cells treated with FF anddiluent; histograms of representative samples are not shown. (n=5samples/condition, representative of 2 experiments). Data is shown asmean-SEM.

FIG. 12B shows the relative basal OCR change of CD8+T cells culturedwith Etomoxir (Eto) in Glu or Gal medium under normoxia (N) or hypoxia(H) normalized to cells cultured with diluent under same condition (setas 100, indicated by dotted line). * above each bar indicate significantdifferences between Eto- and diluent-treated cells. Lines with * aboveshow differences between the connected samples. n=9 samples/condition,representative of 2 experiments. Eto inhibits lipid metabolism.

FIG. 12C shows a bar graph, the effects of FF or Eto on PD-1 expressionchange of CD8+T cells stimulated in Glu (light grey bars) or Gal (darkgrey bars) medium and subjected to hypoxia (H, n=4-6/group,representative of >3 experiments). Data are shown as relative MFI changefor PD-1 stains on cells treated with FF or Eto normalized to the MFIchange of PD-1 stains on cells treated with diluent under the sameconditions (N set at 100, stippled black line). * on top of barsindicates significant differences between cells treated with FF or Etoand with diluent. Histograms of PD-1 expression on representativesamples are not shown.

FIG. 12D shows a bar graph with normalized % change of CD44+CD8+T cellsproducing 3, 2 or 1 factors (from top to bottom of each bar). Functionsof cells treated with FF or Eto are normalized to those of cells treatedwith diluent (set at 300, stippled line; n=5/group, representative of 3assays/group). (*): significant differences of total responses betweencells treated with drug or diluent. * outside of 0: significantdifferences in change of % of 1, 2 and 3 factors (bottom to top).Representative flow plots show levels of factors. All data are shown asmean-SEM.

FIGS. 13A-13B show reducing FA metabolism of activated CD8+T cells undermetabolically challenging conditions in vitro reduces their PD-1expression and effector functions.

FIG. 13A shows normalized change of MFI values for PD-1 stains on PPAR-αKO CD8+T cells cultured under different conditions compared to those ofwt CD8+T cells (n=4-5/condition). * Indicates significant differencebetween wt and PPAR-α KO CD8+T cells. Data are normalized to resultswith wt cells cultured under the same conditions and set at 100.Normoxia, N; Hypoxia, H. All data are shown as mean-SEM.

FIG. 13B shows normalized change of % of PPAR-α KO CD8+T cells producing3, 2 and 1 factors (from top to bottom of each bar) compared to those ofwt CD8+T cells (n=5/condition).

FIGS. 14A-14C show the effect of fenofibrate treatment during in vitrostimulation of OT-1 cells (a cancer model) and the effects on cellfunctions and phenotypes.

FIG. 14A shows the effects of cell function based on % of parentalCD44+CD8+ T cells (Mean-SEM) in a mouse model B14-SIINFEKL using OT-1cells transgenic for SIINFEKL-specific TCR. Measurements are takenindividually for the GzmB, IFN-γ (γ) and TNF-α (α) in control (Co, firstbars under each condition) and FF-treated cells (FF, second bar undereach condition).

FIG. 14B is a graph showing the same measurement on the combination ofthe GzmB, IFN-γ and TNF-α. Control, Co, first bars under each condition;FF-treated cells, FF, second bar under each condition.

FIG. 14C is a graph showing effect on phenotypes, measured as MFI(mean-SEM) for the antigens CD127, CD62L, KLRG1 and PD-1. Control, Co,first bars under each condition; FF-treated cells, FF, second bar undereach condition. These data show that FF treatment operates in cancersother than melanoma, and without administration of a specific vaccineused to induce T cell proliferation.

FIGS. 15A-15F show FF treatment during in vitro stimulation of OT-1cells.

FIG. 15A is a bar graph showing the effect of various concentration ofFF on proliferation.

FIG. 15B is a bar graph showing the effect of various concentration ofFF on PD1.

FIG. 15C is a bar graph showing the effect of various concentration ofFF on Bodipy C16.

FIG. 15D is a bar graph showing the effect of various concentration ofFF on Cpt-a.

FIG. 15E is a bar graph showing the effect of various concentration ofFF on PPARα.

FIG. 15F shows a heat map indicating the various transcripts andfunctions affected by the treatment.

FIG. 16 is a graph showing the in vitro OT-1 CD8⁺ T cellsstimulated/activated with the SIINFEKL peptide and pretreated withfenofibrate can inhibit the growth of melanoma tumor cells (transfectedwith the peptide) in a mouse model. See Example 8. The graph plots tumorvolume (mm³) vs. time (days). The effect of the FF-treated, peptideactivated OT-1 cells (black closed circle) are contrasted withactivated, control treated OT-1 cells (light gray closed circles) andnaïve, unstimulated, untreated OT-1 cells (open circle).

DETAILED DESCRIPTION

The methods and compositions disclosed herein relate to the ability to“switch” the metabolism of T cells from the use of glucose andglycolysis to obtain energy to the use of fatty acid catabolism fordirect or adjunctive treatment of cancer.

The inventors have determined and support via the data presented hereinthe use or supplemental use of metabolic interventions, i.e., drugs,compounds or reagents, that promote fatty acid catabolism by adoptivelytransferred or vaccine-induced CD8+T cells to improve the efficacy ofcancer immunotherapy. Specifically, this invention is based upon thedeterminations that: Metabolic stress within the TME decreases functionsof tumor-infiltrating CD8+TILs. Lack of glucose within the TME forcesCD8+TILs to switch to fatty acid catabolism. CD8+TILs subjected to lowO₂ and glucose gain energy through ketone body catabolism; and CD8+TILsconditioned to increase FA catabolism show improved antitumor activity.

Specifically, as described below in the examples, using a mouse melanomamodel, it was shown that metabolic challenges due to lack of glucose(Glu) combined with hypoxia within the TME impairs vaccine-inducedCD8+TILs functions. When simultaneously subjected to hypoglycemia andhypoxia, CD8+ TILs enhance catabolism of fatty acids (FAs) includingketone bodies, which partially preserves their effector functions.Preconditioning CD8+ TILs to increase FA catabolism further improvestheir ability to slow tumor progression, although PD-1 expressionconcomitantly increases. Blockade of PD-1 signaling also reduces ordelays tumor progression although it fails to affect vaccine-inducedCD8+TIL functions or metabolism. PD-1 blockade (i.e., anti-PD-1treatment) acts synergistically with metabolic reprogramming of T cells,particularly TILs, to achieve superior antitumor efficacy. Thus themethods and compositions provided herein use metabolic interventions toimprove the efficacy of cancer immunotherapy.

The methods and compositions provided herein offer potential therapeuticinterventions to delay loss of TIL functions caused by metabolic stress.First of all, the supporting data shown herein indicates that continuedT cell receptor signaling in vaccine-induced CD8+ TILs is not the solefactor that drives their functional exhaustion, as this fate is alsoencountered by CD8+ TILs directed to an antigen that is not expressedwithin the TME. Additional data show that TILs experience metabolicstress within a glucose- and oxygen-lacking TME, which becomesincreasingly severe during tumor progression. Hypoxia within solidtumors causes CD8+ TILs to increase the hypoxia-induced factor (HIF)-1αsignaling, which drives CD8+ T cell exhaustion by enhancing co-inhibitorlymphocytes activation gene (LAG)-3 expression and reducing the T cells'effector functions.

The data in the examples further show that lack of glucose within theTME enhances expression of PD-1 and impairs the CD8+ TILs' functions. Itforces CD8+ TILs to switch to fatty acid (FA) catabolism demonstrated bystable isotope tracing directly in vivo and liquid chromatography-massspectrophotometry. Promoting FA catabolism of CD8+TILs through the PPARγagonist fenofibrate slightly enhances their PD-1 expression, butnevertheless augments their effector functions and thereby achievesclinical benefits by delaying tumor progression. The data show thathypoglycemia and hypoxia play a critical role in driving metabolicreprogramming and functional impairment of CD8+TILs. They furtherindicate that metabolic interventions that increase FA catabolism byCD8+TILs in a Glu-deprived TME improve the efficacy of cancerimmunotherapy

Thus various methods for treating cancer comprise administering to amammalian subject having a cancer a T cell that is pretreated orconditioned ex vivo or in vitro with a compound or reagent that promotesthe use of fatty acid catabolism rather than glucose for energyproduction by the pre-treated T cells and/or administering a compound orreagent that promotes the use of fatty acid catabolism by tumorantigen-specific T cells in the tumor microenvironment (referred tovariously as “a fatty acid catabolism promoter” or “fatty acidcatabolism-promoting compound”). Further methods involve administering atumor-specific vaccine composition with the pretreated T cells or withthe fatty acid catabolism-promoting compound. All of these possiblemethods that take advantage of switching the energy productionmetabolism of the T cells can optionally be coupled with checkpointinhibition, such as PD-1 blockade.

Certain components and definitions used in the description of thesemethods and compositions are defined below.

“Patient” or “subject” as used herein means a mammalian animal,including a human, a veterinary or farm animal, a domestic animal orpet, and animals normally used for clinical research. More specifically,the subject of these methods and compositions is a human.

As used herein, the term “T cell(s)” or “T cell population” mean anyhuman or mammalian T cell(s). In one embodiment, the T cell or populatedis activated. In one embodiment, the T cell is an autologous orheterologous, naturally occurring T cell. In another embodiment, the Tcell is a recombinantly or synthetically modified T cell construct. Insome embodiments, the T cell to be pretreated is a primary T cell, a CD8(cytotoxic) T cell, a CD8 (cytotoxic) T cell, a T infiltratinglymphocyte (TIL), an NK T cell or another T cell. In one embodiment, theT cell is obtained from the peripheral blood, TME or other fluid of thesame mammalian subject into whom the T cell which is pre-treated orconditioned by the methods described herein is to be administered. Inanother embodiment, the T cell to be pretreated is primary T cell, a CD8(cytotoxic) T cell, or an NK T cell or other T cell obtained from a bonemarrow transplant match for the subject. Other suitable T cells includeT cells obtained from resected tumors, a polyclonal or monoclonaltumor-reactive T cell. In one embodiment, the T cell is obtained byapheresis. In still other embodiments, the T cell is modifiedrecombinantly or synthetically to express a heterologous antigenreceptor. In one embodiment, the T cell is expresses a chimeric antigenreceptor (CAR) or a chimeric endocrine receptor (CER). Such CARs or CERsare described in e.g., Sadelain, M et al, “The basic principles ofchimeric antigen receptor (CAR) design” 2013 April, Cancer Discov. 3(4):388-398; International Patent Application Publications WO2013/044255 andWO2016/054153, US patent application publication No. US 2013/0287748,and other publications directed to the use of such chimeric constructs.These publications are incorporated by reference to provide informationconcerning various components useful in the design of some of theconstructs described herein. Such CAR or CER T cells are geneticallymodified lymphocytes expressing a ligand that allows them to recognizean antigen of choice. Upon antigen recognition, these modified T cellsare activated via signaling domains converting these T cells into potentcell killers. An advantage over endogenous T cells is that they are notMHC restricted, which allows these T cells to overcome an immunesurveillance evasion tactic used in many tumor cells by reducing MHCexpression. In still other embodiment, the T cell for pretreatment is anendogenous or heterologous human T cell or human T cell line. Any T cellmay be subjected to pretreatment ex vivo with a selected fatty acidcatabolism promoter to “switch” its metabolic function from glycolysisto FA catabolism for the purposes of the methods and compositionsprovided herein.

As used herein the term “cancer” refers to or describes thephysiological condition in mammals that is typically characterized byunregulated cell growth. More specifically, as used herein, the term“cancer” means any cancer characterized by the presence of a solidtumor. Suitable cancers for treatment by the methods described herein,include, without limitation, melanoma, breast cancer, brain cancer,colon/rectal cancer, lung cancer, ovarian cancer, adrenal cancer, analcancer, bile duct cancer, bladder cancer, bone cancer, endometrialcancer, esophagus cancer, eye cancer, kidney cancer, laryngeal cancer,liver cancer, head and neck cancer, nasopharyngeal cancer, osteosarcoma,oral cancer, ovarian cancer, pancreatic cancer, prostate cancer,rhabdomosarcoma, salivary gland cancer, stomach cancer, testicularcancer, thyroid cancer, vaginal cancer, lung cancer, and neuroendocrinecancer.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. In one embodiment, the tumor targetedby the methods is characterized by hypoxia, significant infiltrationwith T lymphocytes, and low glucose in the tumor microenvironment.

As used herein, a compound or reagent that promotes the use of fattyacid catabolism by tumor antigen-specific T cells in the tumormicroenvironment (referred to variously as “a fatty acid catabolismpromoter” or “fatty acid catabolism-promoting compound”) includeswithout limitation, compounds such as fenofibrate, clofibrate,gemfibrozil, ciprofibrate, bezafibrate or an AMPK activator, such as5-aminoimidazole-4-carboxamide riboside. Other compounds, small moleculecompounds or peptides, proteins or polypeptides, useful in these methodsmay be identified by one of skill in the art.

As used herein, the term “checkpoint inhibitor” refers to a compositionor composition in the form of an antibody or a small molecule that bindsor inhibits various checkpoint proteins. Such checkpoint proteins,including, without limitation, PD-1, PD-L1, CTLA-4, BTLA and CD160. Asexamples, known checkpoint inhibitors include the antibodies ipilimumab(Yervoy®), pembrolizumab (Keytruda®), and nivolumab (Opdivo®), amongothers. Other checkpoint inhibitors developed as small molecules orother checkpoint binding antibodies or antibody fragments are includedin this definition.

As used herein, the term “antibody” refers to all types ofimmunoglobulins, including IgG, IgM, IgA, IgD, and IgE, includingantibody fragments. The antibody can be monoclonal or polyclonal and canbe of any species of origin, including (for example) mouse, rat, rabbit,horse, goat, sheep, camel, or human, or can be a chimeric antibody. See,e.g., Walker et al., Molec. Immunol. 26:403 (1989). The antibodies canbe recombinant monoclonal antibodies produced according to knownmethods, see, e.g., U.S. Pat. Nos. 4,474,893 or 4,816,567, which areincorporated herein by reference. The antibodies can also be chemicallyconstructed according to known methods, e.g., U.S. Pat. No. 4,676,980which is incorporated herein by reference. See also, U.S. Pat. No.8,613,922, which is incorporated herein by reference. Antibody fragmentsare antigen binding fragments which include, for example, Fab, Fab′,F(ab′)2, and Fv fragments; domain antibodies, bifunctional, diabodies;vaccibodies, linear antibodies; single-chain antibody molecules (scFV);heavy chain or light chain complementarity determining regions, andmultispecific antibodies formed from antibody fragments. Suchantigen-binding fragments can be produced by known techniques.

By “therapeutic reagent” or “regimen” is meant any type of treatmentemployed in the treatment of cancers with or without solid tumors,including, without limitation, chemotherapeutic pharmaceuticals,biological response modifiers, radiation, diet, vitamin therapy, hormonetherapies, gene therapy, surgical resection, etc.

By “an immunotherapeutic composition targeting an antigen or ligand onthe tumor cell” is meant any composition including cancer vaccines thattarget a cancer antigen in order to stimulate the subject's immunesystem. Such immunotherapeutic compositions are designed to elicit ahumoral (e.g., antibody) or cellular (e.g., a cytotoxic T cell or Thelper) response, or, in one embodiment, an innate immune response, ismounted to a target gene product delivered by the immunogeniccomposition following delivery to a mammal or animal subject. In oneembodiment immunotherapeutic compositions useful in these methodsinvolve presentation of the antigen to the subject's immune system viavirus vectors, e.g., adenovirus, adeno-associated virus, lentivirus,retrovirus, poxvirus or others, or via virus-like particles (VLP). Inanother embodiment the immunotherapeutic composition used in the methodsdescribed herein is a DNA or RNA construct that expresses a cancerantigen. In another embodiment the immunotherapeutic composition used inthe methods described herein is a composition comprising cancer antigensor fragments thereof as peptides or proteins. In another embodiment theimmunotherapeutic composition used in the methods described herein is amonoclonal antibody or antigen-binding fragment(s) that specificallybind cancer antigens. The compositions are those that are created usingknown recombinant and synthetic techniques. See, e.g., reference in theexamples to an exemplary melanoma immunotherapeutic composition,AdC68-gDMelapoly described in detail in U.S. Pat. No. 9,402,888 and inFIG. 7 thereof Many immunotherapeutic cancer “vaccine” are known anddescribed in the art that can be used in the methods described herein.

By “antigen or ligand on the tumor cell” is meant a full-length,wild-type tumor-specific antigen or mutated tumor-specific antigens ortumor-associated antigens. Tumor-specific antigens are those epitopesand proteins found on a selected specific cancer or tumor cell, and noton all cancer cells. Cancer-associated antigens are antigens that may beassociated with more than one cancer or tumor cell type. Exemplarycancer-specific antigens can include, without limitation, 707-AP, alpha(a)-fetoprotein, ART-4, BAGE; b-catenin/m, b-catenin/mutated Bcr-abl,CAMEL, CAP-1, mCASP-8, CDC27m, CDK4/m, CEA, CT, Cyp-B, MAGE-B2, MAGE-B1,ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/neu, HPV-E7,HSP70-2M HST-2, hTERT, iCE, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1,MC1R, MUC1, MUM-1, -2, -3, P15, p190 minor bcr-abl. Still other suitabletumor or cancer genes encode VEGFR1, VEGFR2, MAGE-A1, MUC-1, Thymosin(31, EGFR, Her-2/neu, MAGE-3, Survivin, Heparanase 1, Heparanase 2, andCEA, among others. Still other suitable antigens are those listed in thereferences, and incorporated by reference herein. See, also, textsidentifying suitable antigens, such as Scott and Renner, in Encyclopediaof life Sciences 2001 Eds., John Wiley & Sons, Ltd.

By “vector” is meant an entity that delivers a heterologous molecule tocells, either for therapeutic or vaccine purposes. As used herein, avector may include any genetic element including, without limitation,naked DNA, a phage, transposon, cosmid, episome, plasmid, or a virus orbacterium. Vectors are generated using the techniques and sequencesprovided herein, described in the examples, and in conjunction withtechniques known to those of skill in the art. Such techniques includeconventional cloning techniques of cDNA such as those described in textssuch as Green and Sambrook, Molecular Cloning: A Laboratory Manual.4^(th) Edit, Cold Spring Harbor Laboratory Press, 2012, use ofoverlapping oligonucleotide sequences of the Salmonella genomes,polymerase chain reaction, and any suitable method which provides thedesired nucleotide sequence.

By “administering” or “route of administration” is meant delivery of theimmunotherapeutic composition, or the fatty acid catabolism-promoter, orthe checkpoint inhibitor or the pre-treated T cells used in the methodsherein, to the subject. As discussed in detail below, these methods canbe independent for each components of the method. Each administrationmethod can occur with or without a pharmaceutical carrier or excipient,or with or without another chemotherapeutic agent into the TME of thesubject. Conventional and pharmaceutically acceptable routes ofadministration include, but are not limited to, systemic routes, such asintraperitoneal, intravenous, intranasal, intravenous, intramuscular,intratracheal, subcutaneous, and other parenteral routes ofadministration or intratumoral or intranodal administration. In oneembodiment, the route of administration is oral. In another embodiment,the route of administration is intraperitoneal. In another embodiment,the route of administration is intravascular. Routes of administrationmay be combined, if desired. In some embodiments, the administration isrepeated periodically, as discussed in detail below.

In the context of the compositions and methods described herein,reference to “one or more,” “at least five,” etc. of the compositions,compounds or reagents listed means any one or any and all combinationsof the compositions, reagents or compounds listed.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively, i.e., to include otherunspecified components or process steps. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively, i.e., to exclude components or steps notspecifically recited.

As used herein, the term “about” means a variability of 10% from thereference given, unless otherwise specified.

It is to be noted that the term “a” or “an”, refers to one or more, forexample, “an miRNA,” is understood to represent one or more miRNAs. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs and byreference to published texts, which provide one skilled in the art witha general guide to many of the terms used in the present application.

In one aspect of this invention, a composition for adoptive transfer toa mammalian subject comprises a T cell or T cell population that hasbeen pretreated ex vivo or in vitro with a compound or reagent thatconditions the cell to use fatty acid catabolism for energy productionby the T cells. In one embodiment, the compound or reagent that promotesthe use of fatty acid catabolism is fenofibrate. In another embodiment,the compound or reagent that promotes the use of fatty acid catabolismis clofibrate, gemfibrozil, ciprofibrate, bezafibrate, an AMPKactivator, or 5-aminoimidazole-4-carboxamide riboside. Still othermetabolic “switching” reagents are anticipated to be useful in the samemanner.

These compositions may employ as the T cells for such pre-treatment anautologous or heterologous, naturally occurring T cell or arecombinantly or synthetically modified T cell construct. The T cell orpopulation may be a human T cell or natural killer (NK) T cell or Tinfiltrating lymphocyte (TIL) obtained from the subject or from a bonemarrow transplant match for the subject. In still other embodiments theT cell or population is obtained from human peripheral blood or from thetumor microenvironment of the subject. In still other embodiments, the Tcell is modified to express a heterologous antigen receptor, or achimeric antigen receptor (CAR-T) or a chimeric endocrine receptor(CER-T) prior to said pretreatment. In still other embodiments, the Tcell or population slated for pretreatment is an endogenous orheterologous human T cell or human T cell line. In yet otherembodiments, the T cell is a TIL or a CD8+ T cell. These compositionsare prepared for adoptive transfer for the treatment of cancer, with orwithout an accompanying checkpoint inhibitor or tumor antigen specificimmunological composition or vaccine.

In one embodiment of the methods described herein, a method for treatingcancer comprises co-administering to a subject having a cancercharacterized by a solid tumor an immunotherapeutic compositiontargeting an antigen or ligand on the tumor cell; and a compound orreagent that promotes the use of fatty acid catabolism by tumorantigen-specific T cells in the tumor microenvironment. In anotherembodiment, this method also involves co-administering a selectedcheckpoint inhibitor in the form of an antibody or a small molecule.

In another embodiment of the methods described herein, a method fortreating cancer comprises administering to a subject having a cancercharacterized by a solid tumor an immunotherapeutic compositiontargeting an antigen or ligand on the tumor cell; and a selected T cell,e.g., a tumor antigen-specific T cell or CAR, etc, pretreated ex vivowith a compound or reagent that promotes the use of fatty acidcatabolism by the T cells so that the T cell uses fatty acids ratherthan glucose for energy production. These pretreated T cells can then beused for adoptive cell transfer.

In still another embodiment of the methods described herein, a methodfor treating cancer comprises administering to a subject having a cancera composition comprising a T cell pretreated ex vivo or in vitro with acompound or reagent that promotes or switches the cell from usingglucose for energy production to using fatty acids and FA catabolism forenergy production. The T cells for such pre-treatment are selected fromthe list of T cells identified above.

By “pre-treatment with a selected fatty acid catabolism promoter” ismeant that the selected T cell is cultured and expanded in the presenceof the selected fatty acid catabolism promoter, e.g., fenofibrate atbetween about 1 to about 500 μM, for the entire or a fraction of thetime of T cell expansion to condition the T cell to use fatty acidsrather than glucose for energy production. In one embodiment a suitableconcentration of the fenofibrate (or similar fatty acid catabolismpromoter) is at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, to at least about500 μM. Similarly intervening concentration between any two numberslisted is encompassed in the term “suitable concentration). The time ofT cell expansion in one embodiment means the entire time of in vitroculture, which can span several hours to at least several days. Inanother embodiment, the time of T cell expansion is minimally 24 hoursof in vitro culture. Other time periods for pre-treatment with the fattyacid catabolism promoter may be at least 1, 5, 10, 15, or 20 or morehours, or any intervening times between any specified number of hoursstated herein. The pretreated T cells are then administered to thesubject by well-known adaptive cell transfer techniques.

In one embodiment, the pretreated T cells are administered to treatcancer as a single therapy. In another embodiment, this method involvesco-administering the immunotherapeutic compositions with the pretreatedT cell. In still other embodiments, the method can also includeadministering a checkpoint inhibitor in the form of an antibody or asmall molecule either simultaneously with or sequentially with thepretreated cells and/or the immunotherapeutic composition.

In still another variation of the method for treating cancer, theco-administration includes the immunotherapeutic composition, the fattyacid catabolism promoter (i.e., administered as a compound) and theselected pretreated T cells identified herein. In another embodiment,this method also involves co-administering a checkpoint inhibitor in theform of an antibody or a small molecule.

In any of the methods described herein, the immunotherapeuticcomposition and the fatty acid catabolism-promoting compound or reagent,or the immunotherapeutic composition and the pretreated T cell areadministered substantially simultaneously. In another embodiment, theimmunotherapeutic composition and the fatty acid catabolism-promotingcompound or reagent, or the immunotherapeutic composition and thepretreated T cell are administered sequentially by the same or differentroutes of administration. The routes of administration selected dependupon the nature of the compositions. For example, if the fatty acidcatabolism promoter is fenofibrate or another small chemical molecule,such molecules may be administered orally in doses known and acceptedfor other pharmaceutical uses of these drugs. In one embodiment, theimmunotherapeutic composition and fatty acid catabolism promoter areindependently administered systemically by intramuscular,intraperitoneal, intravenous, intratumoral or intranodal administration.In another embodiment, composition (b) is administered orally.

In other administration protocols, the fatty acid catabolism-promotingcompound or reagent or the pretreated T cells are administered once orrepeatedly from at least one to 14 days. In some protocols theadministration occurs one to 14 days after administration of theimmunotherapeutic composition. In certain embodiments, theimmunotherapeutic composition is administered in a single dose. In otherembodiments, the immunotherapeutic composition is administered as abooster dose.

In still further aspects of these methods, the subject may be treatedwith other anti-cancer therapies before, during or after treatment withthe pre-treated T cells alone or with the immunotherapeutic compositionand the fatty acid catabolism promoter or with the combination of theimmunotherapeutic compositions and pre-treated cells. Such treatment maybe concurrent or simultaneous with the fatty acid catabolism promoter oroverlap treatment with the modified T cells adoptive transfer and/or thecheckpoint inhibitors. In one embodiment, the methods involve treatingthe subject with chemotherapy before administering the immunotherapeuticcomposition and/or the fatty acid catabolism promoter. In still anotherembodiment, the method further comprises depleting the subject oflymphocytes and optionally surgically resecting the tumor prior toadoptive transfer of the selected T cells pretreated ex vivo with anfatty acid catabolism promoter to condition the T cell to use fattyacids rather than glucose for energy production.

In some embodiments, the pretreated cells are administered in a singledose, followed by optional administration of a checkpoint inhibitor.These doses may be repeated. In yet other embodiments of the methods,the immunotherapeutic composition is administered in a single dosewithout any booster, followed by administration of at least one of thefatty acid catabolism promoter, the selected pre-treated T cells, and/orthe checkpoint inhibitors. In yet another embodiment, theimmunotherapeutic composition is re-administered as a booster dosefollowing administration of the fatty acid catabolism promoter, theselected pre-treated T cells, and/or the checkpoint inhibitors.

Any of these therapeutic compositions and components of the methods maybe administered to a patient, preferably suspended in a biologicallycompatible solution or pharmaceutically acceptable delivery vehicle. Thevarious components of the methods are prepared for administration bybeing suspended or dissolved in a pharmaceutically or physiologicallyacceptable carrier such as isotonic saline; isotonic salts solution orother formulations that will be apparent to those skilled in suchadministration. The appropriate carrier will be evident to those skilledin the art and will depend in large part upon the route ofadministration. Other aqueous and non-aqueous isotonic sterile injectionsolutions and aqueous and non-aqueous sterile suspensions known to bepharmaceutically acceptable carriers and well known to those of skill inthe art may be employed for this purpose.

Dosages of these therapeutic compositions will depend primarily onfactors such as type of composition (i.e., selected pre-treated T cells,vectors, nucleic acid constructs or proteins) the condition beingtreated, the age, weight and health of the patient, and may thus varyamong patients. The dosages for administration of the components of themethods are the conventional dosages known to be useful foradministering that component. An attending physician may selectappropriate dosages using the following as guidelines.

In one embodiment, a useful dosage of a pre-treated T cell is asingle-infusion maximum tolerated dose (MTD), which may be determined bydose escalation studies in animal models. In one embodiment, a typicalefficacious and non-toxic dose of T cells is between about 2×10⁴ to5×10⁹ cells per kg/subject body weight. Other doses, such as 10⁵ or 10⁶or 10⁷ or 10⁸ can be useful. See, the methods for dose determination asdescribed in e.g., WO2016/054153 and in other CAR publications in theart.

In one embodiment, a typical dosage of an immunotherapeutic compositiondepends upon the nature of the composition. For example, if thecomposition is delivered in a viral vector, a therapeutically effectiveadult human or veterinary dosage of a viral vector is generally in therange of from about 100 μL to about 100 mL of a carrier containingconcentrations of from about 1×10⁶ to about 1×10¹⁵ particles, about1×10¹¹ to 1×10¹³ particles, or about 1×10⁹ to 1×10¹² particles virus.

If the composition (e.g., the immunotherapeutic composition, fatty acidcatabolism-promoting compound or checkpoint inhibitor) is administeredas an antibody or other protein, the dosages may range between a unitdosage of between 0.01 mg to 100 mg of protein (which is equivalent toabout 12.5 μg/kg body weight). The dosage of the checkpoint inhibitormay be adjusted based on known toxicities of the particular antibody orsmall molecule used.

If any of the immunotherapeutic composition or the other components ofthe method is administered as naked DNA, the dosages may range fromabout 50 μg to about 1 mg of DNA per mL of a sterile solution.

Similarly, the doses of the fatty acid catabolism promoting compound maybe similar to those administered for other uses, e.g., for cholesterolcontrol or hyperlipidemia, of the similar compound. For example, FF maybe administered at dosages of from 40 mg/day to 120 mg/day for adults.In yet another embodiment, a “standard” efficacious and non-toxic doseof pretreated T cells for adoptive transfer is about 10⁷ cells. Asanother example, the number of adoptively transferred T cells can beoptimized by one of skill in the art. In one embodiment, such a dosagecan range from about 10⁵ to about 10¹¹ cells per kilogram of body weightof the subject. Other dosages are taught in the references recitedherein and can be readily adjusted by one of skill in the art dependingupon the treatment regimen, physical condition of the patient, type andstage and location of the tumor being treated, and taking intoconsideration other ancillary chemotherapies being used to treat thepatient.

In yet another aspect, a therapeutic regimen is provided for thetreatment of cancer comprising administering to a subject having acancer characterized by a solid tumor a single dose of animmunotherapeutic composition targeting an antigen or ligand on thetumor cell on a day 1 of treatment. In this regimen, the subject isthereafter administered a compound or reagent that promotes the use offatty acid catabolism by tumor antigen-specific T cells in the tumormicroenvironment. The first dose of the fatty acid catabolism promotingcompound of reagent begins on any of day 0, 1, 2, 3, 4 or 5 oftreatment. Also involved in this regimen is the step of administeringthe fatty acid catabolism promoting compound or reagent daily from thebeginning day of treatment of immunotherapeutic composition until a dayoccurring between day 7 to day 30 of treatment. The checkpointinhibitors may be administered at the same time or following theadministration of the fatty acid catabolism promoting compound.

In yet another aspect, a therapeutic regimen is provided for thetreatment of cancer comprising administering to a subject having acancer characterized by a solid tumor a single dose of animmunotherapeutic composition targeting an antigen or ligand on thetumor cell on a day 1 of treatment. In this regimen, the subject isthereafter administered by adoptive transfer the selected T cellspretreated ex vivo with a compound or reagent that promotes the use offatty acid catabolism by tumor antigen-specific T cells to condition theT cell to use fatty acids rather than glucose for energy production. Theadoptive transfer of the pre-treated T cells conditioned with the fattyacid catabolism promoting compound can occur on any of day 0-14 afteradministration of the immunotherapeutic composition. Thus in certainembodiments, the adoptive transfer occurs on day 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13 or day 14 or even later than day 14, afterimmunotherapeutic composition administration. Other dates may beselected depending upon how long the immunotherapeutic composition isexpressed in vivo. This expression depends upon the type of vaccine andthus the timing of co-administration in the therapeutic regimen may beadjusted by one of skill in the art. The checkpoint inhibitors may beadministered at the same time or following the administration of thepre-treated T cells.

In yet another embodiment, the discoveries of the inventors also permitsa method of enhancing the survival of a chimeric antigen receptor-T cellor a chimeric endocrine receptor-T cell or ex vivo expanded tumorantigen-specific T cells, such as those described⁴¹ and in e.g.,International patent application publication No. WO2012/079000 andInternational patent application No. PCT/US2015/053128, eachincorporated by reference. In one embodiment, the pretreated T cell isobtained from peripheral blood and modified to express a chimericantigen receptor or a chimeric endocrine receptor and pretreated ex vivowith the fatty acid catabolism promoting compound or reagent. Thepretreated T cell is an endogenous or heterologous human T cell or humanT cell line. The pretreated T cell is a CD8+ T cell. In this method, theT cell(s) are pretreated ex vivo with a compound or reagent thatpromotes the use of fatty acid catabolism for energy production by tumorantigen-specific T cells in the tumor microenvironment as discussedabove and then administered to the patient having a solid tumor byadoptive cell transfer, as described in the incorporated references.

The rationale for these methods is based in the inventors' observationsin a mouse melanoma model that bystander CD8+TILs lose effectorfunctions and increase expression of co-inhibitors. The data show thatmetabolic stress within the tumor microenvironment (TME) affectsdifferentiation and effector functions of CD8⁺TILs in the mouse melanomamodel. Hypoxia through HIF-1α and lack of glucose (Glu) enhanceexpression of co-inhibitors and impair CD8+T cell functions. Whensimultaneously subjected to hypoxia and hypoglycemia, CD8+T cellsenhance catabolism of fatty acids (FAs) including ketone bodies.CD8+TILs conditioned to increase FA catabolism augment PD-1 expressionand show improved production of effector molecules.

Hypoxia triggered by suboptimal neoangiogenesis or defects in perfusionwithin solid tumors causes CD8+T cells to increase expression of thehypoxia-induced factor (HIF)-1α and lymphocytes activation gene (LAG)-3and to lose functions. As shown with vaccine-induced CD8⁺TILs and invitro activated polyclonal CD8⁺T cells, hypoxia through hypoxia-inducedfactor (HIF)-1α increases co-inhibitor LAG-3 expression and impairsCD8⁺T cell functions. Limited Glu supply enhances PD-1 expression,reduces effector functions and increases FA catabolism of CD8⁺T cells,which is further enhanced under hypoxia. The inventors showed in theexamples below that CD8⁺TILs in late stage tumors increasingly depend onFA catabolism fueled by FA uptake and triacylglycerol (TG) turnover tomeet their energy demand, which increases PD-1 expression but preservessome effector functions. Promoting FA catabolism of CD8⁺TILs improvestheir antitumor efficacy. Further, LAG-3 overexpression and functionalimpairments can be reversed by genetic knock-down of HIF-1α.

Lack of glucose, which due to its consumption by tumor cells becomesscarce within a TME, impairs the CD8+TILs' functions, enhancesexpression of PD-1 and forces cells to switch to fatty acid metabolismas was shown by liquid chromatography-mass spectrophotometry and stableisotope tracing. This metabolic preference is further enhanced underhypoxia. Energy production through fatty acid oxidization rather thanglucose requires more O₂ to generate equivalent amounts of ATP, whichmay not be sustainable under hypoxia. Ketone bodies, byproduct of fattyacid oxidization, are highly efficient fuels that require less O₂. Also,as supported by the data in the examples, and shown previously for cellsof the nervous system subjected to hypoxia and hypoglycemia, CD8+TILsonce they enter areas of hypoxia within a tumor switch to energyproduction through ketone body catabolism.

In recent years blockade of immunological checkpoints has evolved as oneof the most promising therapies to enhance tumor antigen-specific immuneresponses and achieved durable clinical responses in cancer patients.Treatments with immune checkpoint inhibitors partially rescue TILfunctions and have yielded promising results in cancer patients.⁴ Theassumption has been that antibodies, which inhibit signaling throughimmunoinhibitors, such as programmed cell death protein (PD)-1, preservefunctions of T cells that due to chronic antigen stimulationdifferentiate towards exhaustion. The data provided herein show thatcontinued T cell receptor signaling in vaccine-induced CD8+ tumorinfiltrating T cells (TILs) is not the sole factor that drives theirexhaustion and functional failure as this fate is also encountered byCD8+ TILs directed to an antigen that is not expressed within the TME.Additional data gained with vaccine-induced TILs in comparison to CD8+ Tcells stimulated under various culture conditions in vitro show thatTILs experience metabolic stress within a glucose- and oxygen-lackingTME, which becomes increasingly severe during tumor progression.

Overall these data as presented in the Examples support that fatty acidmetabolism is essential for CD8+TILs to preserve their tumoricidalfunctions within the TME. Additional data using drugs that promote fattyacid oxidization or mice with genetic alteration that affect lipidmetabolism furthermore show that tumor antigen-specific CD8+T cellsconditioned during activation to use fatty acids rather than glucose forenergy production show better preserved functions within the TME andachieve longer delays in tumor progression although they express higherlevels of PD-1.

EMBODIMENTS OF THE INVENTION

Various embodiments of the invention include the following:

a) A method for treating cancer comprising administering to a subjecthaving a cancer a T cell or T cell population that is pretreated orconditioned ex vivo or in vitro with a compound or reagent that promotesthe use of fatty acid catabolism rather than glucose for energyproduction by the pre-treated T cells;

b) A method for treating cancer comprising co-administering to a subjecthaving a cancer: an immunotherapeutic composition targeting an antigenor ligand on a tumor cell in the subject with one or more of (i) acompound or reagent that promotes the use of fatty acid catabolism bytumor antigen-specific T cells in the tumor microenvironment; and (ii) aT cell pretreated ex vivo with (i) to condition the T cell to use fattyacids rather than glucose for energy production for adoptive celltransfer;

c) Either of the methods above, further comprising administering acheckpoint inhibitor in the form of an antibody or a small molecule;

d) Any of the methods herein, wherein the checkpoint inhibitor is ananti-PD-1 antibody or small molecule ligand;

e) Any of the methods herein, wherein the cancer is characterized by thepresence in the subject of a solid tumor;

f) Any of the methods herein, wherein the cancer is melanoma, breastcancer, brain cancer, colon/rectal cancer, lung cancer, ovarian cancer,adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bonecancer, endometrial cancer, esophagus cancer, eye cancer, kidney cancer,laryngeal cancer, liver cancer, head and neck cancer, nasopharyngealcancer, osteosarcoma, oral cancer, ovarian cancer, pancreatic cancer,prostate cancer, rhabdomosarcoma, salivary gland tumors, stomach cancer,testicular cancer, thyroid cancer, vaginal cancer, neuroendocrinecancer;

g) Any of the methods herein, wherein the compound or reagent thatpromotes the use of fatty acid catabolism by T cells is fenofibrate;

h) Any of the methods herein, wherein the compound or reagent thatpromotes the use of fatty acid catabolism by T cells is clofibrate,gemfibrozil, ciprofibrate, bezafibrate, an AMPK activator or5-aminoimidazole-4-carboxamide riboside;

i) Any of the methods herein, wherein the T cell is an autologous orheterologous, naturally occurring T cell or a recombinantly orsynthetically modified T cell construct;

j) Any of the methods herein, wherein the T cell is a human T cell ornatural killer (NK) T cell or T infiltrating lymphocyte (TIL) obtainedfrom the subject or from a bone marrow transplant match for the subject;

k) Any of the methods herein, wherein the T cell is obtained from humanperipheral blood or from the tumor microenvironment of the subject;

l) Any of the methods herein, wherein the T cell is modified to expressa heterologous antigen receptor, or a chimeric antigen receptor or achimeric endocrine receptor prior to said pretreatment;

m) Any of the methods herein, wherein the T cell is an endogenous orheterologous human T cell or human T cell line;

n) Any of the methods herein, wherein the T cell is a CD8+ T cell;

o) Any of the methods herein, wherein said immunotherapeutic composition(a) is a recombinant virus or virus-like particle that expresses acancer antigen, a DNA construct that expresses a cancer antigen, acomposition comprising cancer antigens or fragments thereof as peptidesor proteins, monoclonal antibodies or antigen-binding fragments thatspecifically bind cancer antigens;

p) Any of the methods herein, wherein the immunotherapeutic compositionand the fatty acid catabolism-promoting compound or reagent, or theimmunotherapeutic composition and the pretreated T cell are administeredsubstantially simultaneously;

q) Any of the methods herein, wherein the fatty acidcatabolism-promoting compound or reagent or the pretreated T cells areadministered once or repeatedly from at least one to 14 days afteradministration of the immunotherapeutic composition;

r) Any of the methods herein, wherein the immunotherapeutic compositionis administered in a single dose or as one or more booster doses;

s) Any of the methods herein, wherein each composition is independentlyadministered systemically by intramuscular, intraperitoneal,intravenous, intratumoral or intranodal administration;

t) Any of the methods herein, wherein the compound or reagent thatpromotes the use of fatty acid catabolism by tumor antigen-specific Tcells in the tumor microenvironment is administered orally;

u) Any of the methods herein, wherein the pretreated T cells areadministered once or repeatedly;

v) Any of the methods herein, wherein the pretreated T cells areadministered in a single dose or as one or more doses;

w) Any of the methods herein, wherein the pretreated T cells areadministered systemically by intravenous injection or infusion;

x) Any of the methods herein, further comprising treating the subjectwith other anti-cancer therapies;

y) Any of the methods herein, further comprising treating the subjectwith chemotherapy before administering the pre-treated T cells,immunogenic composition or compound or reagent that promotes the use offatty acid catabolism;

z) Any of the methods herein, further comprising depleting the subjectof lymphocytes and optionally surgically resecting the tumor prior toadministration of the pretreated T cells;

aa) Any of the methods herein, wherein the tumor targeted by the methodis characterized by hypoxia, significant infiltration with Tlymphocytes, and low glucose in the tumor microenvironment;

bb) A method of modifying a T cell comprising pretreating the T cell exvivo or in vitro with a compound or reagent that conditions the cell touse fatty acid catabolism for energy production by the T cells;

cc) A method of enhancing the survival of a chimeric antigen receptor-Tcell or a chimeric endocrine receptor-T cell or an ex vivo expandedtumor antigen-specific T cells comprising pretreating the T cell ex vivowith a compound or reagent that promotes the use of fatty acidcatabolism for energy production by tumor antigen-specific T cells inthe tumor microenvironment before adoptive cell transfer to a subjecthaving a solid tumor;

dd) Any of the two preceding methods, wherein the compound or reagentthat promotes the use of fatty acid catabolism is fenofibrate;

ee) Any of the three preceding methods herein, wherein the compound orreagent that promotes the use of fatty acid catabolism is clofibrate,gemfibrozil, ciprofibrate, bezafibrate, an AMPK activator, or5-aminoimidazole-4-carboxamide riboside;

ff) Any of the four preceding methods herein, wherein the T cell is anautologous or heterologous, naturally occurring T cell or arecombinantly or synthetically modified T cell construct;

gg) Any of the five preceding methods herein, wherein the T cell is ahuman T cell or natural killer (NK) T cell or T infiltrating lymphocyte(TIL) obtained from the subject or from a bone marrow transplant matchfor the subject;

hh) Any of the six preceding methods herein, wherein the T cell isobtained from human peripheral blood or from the tumor microenvironmentof the subject;

ii) Any of the preceding methods herein, wherein the T cell is modifiedto express a heterologous antigen receptor, or a chimeric antigenreceptor or a chimeric endocrine receptor prior to said pretreatment;The method according to any of claim 28 or 29, wherein the T cell is anendogenous or heterologous human T cell or human T cell line;

jj) Any of the preceding methods herein, wherein the T cell is a CD8+ Tcell;

kk) A therapeutic regimen for the treatment of cancer comprising:

-   -   1. administering to a subject having a cancer characterized by a        solid tumor a single dose of an immunotherapeutic composition        targeting an antigen or ligand on the tumor cell on a day 1 of        treatment;    -   2. administering to said subject a compound or reagent that        promotes the use of fatty acid catabolism by tumor        antigen-specific T cells in the tumor microenvironment, said        first dose of the fatty acid catabolism-promoting compound of        reagent beginning on day 0-5 of treatment;    -   3. administering the fatty acid catabolism-promoting compound or        reagent daily from the beginning day of treatment of (2) until a        day occurring between day 7 to day 30 of treatment;

ll) A composition for adoptive transfer to a mammalian subjectcomprising a T cell that has been pretreated ex vivo or in vitro with acompound or reagent that conditions the cell to use fatty acidcatabolism for energy production by the T cells;

mm) The preceding composition, wherein the compound or reagent thatpromotes the use of fatty acid catabolism is fenofibrate;

nn) The preceding compositions, wherein the compound or reagent thatpromotes the use of fatty acid catabolism is clofibrate, gemfibrozil,ciprofibrate, bezafibrate, an AMPK activator, or5-aminoimidazole-4-carboxamide riboside;

oo) Any of the preceding compositions, wherein the T cell is anautologous or heterologous, naturally occurring T cell or arecombinantly or synthetically modified T cell construct;

pp) Any of the preceding compositions, wherein the T cell is a human Tcell or natural killer (NK) T cell or T infiltrating lymphocyte (TIL)obtained from the subject or from a bone marrow transplant match for thesubject;

qq) Any of the preceding compositions, wherein the T cell is obtainedfrom human peripheral blood or from the tumor microenvironment of thesubject;

rr) Any of the preceding compositions, wherein the T cell is modified toexpress a heterologous antigen receptor, or a chimeric antigen receptoror a chimeric endocrine receptor prior to said pretreatment;

ss) Any of the preceding compositions, wherein the T cell is anendogenous or heterologous human T cell or human T cell line; and

tt) Any of the preceding compositions, wherein the T cell is a CD8+ Tcell

Within the tumor microenvironment vaccine-induced CD8+T cells encountermetabolic stress due to lack of glucose and O₂, which results inincreased expression of co-inhibitors and loss of functions. CD8+ tumorinfiltrating T cells (TILs) react by enhancing catabolism of fatty acidsincluding ketone bodies. Drug-induced increases in fatty acidoxidization further augment expression of the co-inhibitor PD-1 onCD8+TILs but significantly improve the T cells' ability to slow tumorprogression.

The inventors determined that lack of Glu and O₂ plays a critical rolein driving the metabolic reprogramming and functional exhaustion ofCD8⁺TILs. They further indicate that metabolic interventions improve theefficacy of cancer immunotherapy.

The following examples are provided for the purpose of illustration onlyand the invention should in no way be construed as being limited tothese examples but rather should be construed to encompass any and allvariations that become evident as a result of the teaching providedherein. In summary, data presented herein elucidate underlying causes offailures of active cancer immunotherapy using a mouse melanoma model.Melanoma-bearing mice were immunized with a mixture of vaccines thatinduce CD8⁺T cells specific for melanoma-associated antigens (MAAs) andan unrelated tumor antigen (TA), i.e., E7 of human papilloma virus(HPV)-16. Both MAA- and bystander E7-specific CD8⁺TILs increaseco-inhibitor expression and lose functions, contesting the notion thathigh and sustained antigenic stimulation is solely liable forTILexhaustion⁵, although it may contribute by increasing the energydemand of CD8⁺T cells that encounter their cognate antigen. Both CD8⁺TILsubsets increasingly experience metabolic stress due to restricted O₂and glucose supply during tumor progression.

Example 1: Materials and Methods

Cell Lines and Construction of Recombinant Adenovirus and Lentivectors.

The B16 cell line and the vaccines have been describedpreviously.^(40,15) The B16Braf_(V600E) cell line (kindly provided byDr. M Herlyn, Wistar Institute, Philadelphia, Pa.) was derived fromB16.F10 cells by transduction with the lentivector pLU-EF1a-mCherryexpressing mouse Braf_(V600E). HEK 293 cells were used to propagatevaccine vectors. Cells were grown in Dulbecco's modified eagles medium(DMEM) supplemented with 10% fetal bovine serum (FBS).

Molecular construction, rescue, purification and titration ofAdC68-gDMelapoly and AdC68-gDE7 vectors have been described⁴⁰. TheMelapoly transgene sequence (see FIG. 7 of U.S. Pat. No. 9,402,888) iscomposed of an ER signal sequence (ER ss) followed by a pan DR epitope(PADRE), three CD4+T cell epitopes from human (h) Trp-2, and eight CD8+Tcell epitopes from human (h) or mouse (m)Trp-2, mTrp-1, hgp100 andmBrafV600E fused into herpes simplex virus (HSV) glycoprotein (g)D. Thedominant CD8+T response elicited by the AdC68-gDMelapoly vector isdirected against the Trp-1455 epitope (˜90% of MAA-specific CD8+T cellresponse). Trp-1455-tetramer+CD8+T cells were analyzed for phenotypicalstudies while MAA-specific CD8+T cells were assessed for functionalassays by intracellular cytokine staining throughout the figures.

Briefly, gDMelapoly or gDE7 construct was inserted into E1-deleted AdC68viral molecular clone using I-CeuI and PI-SceI sites. The constructedplasmids were used to transfect HEK 293 T cells by calcium phosphate(Invitrogen, Carlsbad, Calif.). Cells containing adenoviral vectors wereharvested 7-10 days later upon plague formation. Virus was furtherpropagated on HEK 293 cells by serial infection and harvested by threecycles of freeze-thawing. Cell-free supernatant from the third cycle ofthawing was used for virus purification by Cesium chloride densityultracentrifugation.

For production of lentivectors, five pLKO.1 lentivectors containingshort hairpin RNAs (shRNAs) targeting different regions of HIF-1α orcontrol RNA were obtained from The RNAi Consortium. The selection markerThy1.1 was cloned from the pLKPO-Thy1.1 lentivector (Addgene) into eachof the shRNA lentivectors. Lentivectors were generated using standardprocedures. The 2^(nd) generation lentivector package system (Addgene)was used and HEK 293T cells were transfected with the packaging plasmidPsPAX2, the envelope plasmid PMD2.G and each of theshRNA-Thy1.1-expressing insert plasmids at a ratio of 3:1:1.Supernatants were collected 48 and 72 hours post transfection.Lentivectors were concentrated by ultracentrifugation at 20,000 rpm, 4°C. for 2 hours. Vector pellets were incubated with PBS on ice for atleast 2 hours before resuspension. The lentivector that showed the mostpronounced reduction of HIF-1α transcripts in transfected cells was usedfor further studies.

Animal Experiments

Female C57Bl/6, B6.SJL-Ptprc^(a)Pepc^(b)/BoyJ (B6 CD45.1⁺),B6.PL-Thy1^(a)/CyJ (B6 CD90.1⁺) and B6; 129S4-Ppara^(tm1Gonz)/J (B6PPAR-α KO) mice (6-8 weeks) were purchased from the National CancerInstitute (NCI) or the Jackson Laboratories and housed at the WistarInstitute Animal Facility. Procedures were conducted following approvedprotocols.

Groups of 5-80 C57BL/6 mice were vaccinated intramuscularly (i.m.) withAdC68 vectors (10¹⁰ virus particles (vp) for AdC68-gDMelapoly; and 10¹¹vp for AdC68-gDE7) diluted in PBS. B16Braf_(V600E) cells (5×10⁴cells/mouse) diluted in phosphate buffered saline (PBS) were inoculatedsubcutaneously (s.c.) into the right flank. Tumor growth was monitoredby measuring the perpendicular diameters of tumors every two days.Depending on size early stage tumors were harvested 10-14 days afterchallenge (referred to as 2 weeks) while late stage tumors wereharvested 4-5 weeks after challenge (referred to as 1 month). Mice wereeuthanized once tumors exceeded a diameter of 1-1.5 cm.

For in vivo treatment, fenofibrate (FF; at 100 mg/kg/day, Sigma) wasfirst diluted in dimethylsulfoxide (DMSO) and then further diluted inPBS and given by oral gavage daily for 3 weeks. Control mice receiveddiluent at the same volume. For adoptive transfer experiments, 1×10⁷ invitro activated CD8⁺T cells transduced with lentivectors or splenocytesfrom vaccinated mice treated with drugs and containing 5×10⁴ Trp-1₄₅₅tetramer⁺CD8⁺T cells per dose were injected intravenously into recipientmice. For FF/control treated splenocytes or wild type/PPAR-α KOsplenocytes co-transfer experiments, splenocytes containing 10⁵ Trp-1₄₅₅tetramer⁺CD8⁺ T cells from each group were mixed and transferred intoCD90.1⁺ recipient mice intravenously.

For PD-1 blockade experiments in NSG or unvaccinated C57BL/6 mice,anti-PD-1 antibody (clone 29F.1A12) or isotype control antibody (Clone:LTF-2, Bio X Cell) were given starting day 3 after tumor challenge. Invaccinated C57BL/6 mice, anti-PD-1 or isotype control antibody treatmentwas started 10 days after vaccination. The antibody was given byintraperitoneal injection every 3rd day at a dose of 200 m g/mouse.

In Vitro Stimulation of CD8⁺ T Cells and Drug Treatments.

Enriched CD8+T cells were activated for 4 days in 6-well platespre-coated with antibodies to CD3 (5 μg/ml) and CD28 (5 μg/mL) (BDBioscience). For some samples, cells were transferred for the last 16hours to a hypoxia chamber. To study the impact of hypoxia on relativelyresting CD8+T cells, enriched CD8+T cells were stimulated for 48 hoursunder normoxia. Cells were then removed from the plates, washed andreplated in fresh medium with 100 U/ml human IL-2 for 96 hours, followedby culture in normoxia or hypoxia with IL-2 for another 36 hours beforeanalysis. Cells were cultured in Roswell Park Memorial Institute (RPMI)medium without Glu (Life Technologies) supplemented with Glu (10 mM) orGal (10 mM), 10% dialyzed FBS (Life Technologies), 20 mM HEPES, 2 mMGlutamax, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol and 1%penicillin-streptomycin. Hypoxia experiments were performed in a ThermoNapco series 8000WJ CO₂ incubator equipped with nitrogen tank for O₂replacement. O₂ level was kept at 1% during CD8+T cells hypoxia culturefor time periods indicated in each assay. In all assays cell viabilitywas assessed before staining. Activated CD8+T Cells cultured in Glumedium under hypoxia showed stable percentage of live cells compared tothose cultured under normoxia (˜70-80% of blasts). Activated CD8+T Cellscultured in Gal medium under normoxia showed reduced viability (˜30-35%of blasts). The frequencies of live cells slightly increased if cellswere subjected to both hypoxia and Gal medium (˜46-50% of blasts). Drugsand corresponding vehicle controls were added as follows:2-deoxy-D-glucose (2-DG, 2 mM, Sigma) or Fenofibrate (FF, 50 μM, Sigma)for the entire culture period; Etomoxir (Eto, 200 μM, Sigma) for thelast 48 hours. DMSO concentrations were kept below 0.2% for all cultureconditions.

CD8⁺T cells from spleens of naive C57Bl/6 mice were purified by negativeselection using magnetic beads (MACS, STEMCELL Technologies). EnrichedCD8⁺T cells were activated for 4 days in 6-well plates pre-coated withantibodies to CD3 (5 μg/ml) and CD28 (5 μg/mL) (BD Bioscience). For somesamples, cells were transferred for the last 16 hours to a hypoxiachamber. To study the impact of hypoxia on resting CD8⁺T cells, purifiedenriched CD8⁺T cells were stimulated for 48 hours under normoxia. Cellswere then washed off the plates and replated in fresh medium with 100U/ml human IL-2 for 96 hours, followed by culture in normoxia or hypoxiawith IL-2 for another 36 hours before analysis. Cells were cultured inRoswell Park Memorial Institute (RPMI) medium without Glu (LifeTechnologies) supplemented with Glu (10 mM) or Gal (10 mM), 10% dialyzedFBS (Life Technologies), 20 mM HEPES, 2 mM Glutamax, 1 mM sodiumpyruvate, 0.05 mM 2-mercaptoethanol and 1% penicillin-streptomycin.Hypoxia experiments were performed in a Thermo Napco series 8000WJ CO₂incubator equipped with nitrogen tank for O₂ replacement. O₂ level waskept at 1% during CD8⁺T cells hypoxia culture for time periods indicatedin each assay. Drugs and corresponding vehicle controls were added asfollows: 2-deoxy-D-glucose (2-DG, 2 mM, Sigma) or Fenofibrate (FF, 50μM, Sigma) for the entire culture period; Etomoxir (Eto, 200 μM, Sigma),Amidepsine A (AmA, 20 μM, Santa Cruz), or Orlistat (OS, 100 μM, Sigma)for the last 48 hours. DMSO concentrations were kept below 0.2% for allculture conditions.

Metabolomics.

For glucose tracing in vitro, cells were stimulated with anti-CD3/CD28for 4 days in medium containing 10 mM ¹³C₆-glucose or ¹³C₆-galactose.For FA tracing in vitro, cells were stimulated for 4 days in regular Gluor Gal medium and switched for the last 4 hours of culture to mediumcontaining 10% delipidated FBS and 400 μM ¹³C₁₆-palmitate-BSA. In vivotracing was conducted by injecting 2 g/kg [U-¹³C] glucose i.p. 30minutes before euthanasia or by feeding ¹³C₁₆-palmitate at 0.5 g/kg 2hours, and injecting¹³C₁₆-palmitate at 150 mg/kg dissolved inintralipid, 20% i.v. 1 hour before euthanasia. Samples were analyzed byLC-MS.

Extracellular Flux Analysis and Fatty Acid Catabolism Assay.

OCR and ECAR for CD8⁺T cells stimulated under different conditions weremeasured with XF24 and XF96 Extracellular Flux Analyzers (SeahorseBioscience) following the manufacturer's instructions.

Hypoxia samples were prepared in a hypoxia chamber under 1% O₂. Deadcells were removed by dead cell removal kit using MACS and live cellswere pre-incubated with 100 μM cobalt chloride before being removed fromthe hypoxia chamber and entered into the Seahorse analyzer. Inexperiments to determine the contribution of fatty acid oxidation (FAO)to OCR, 200 μM ETO was added 15 minutes before the Seahorse analysis.Briefly after repeated measures of basal respiration and lactateproduction, 1 μM OM was added to measure ATP leakage by OCR andglycolytic capacity by ECAR. 1.5 μM FCCP was then added to measuremaximal respiration by OCR followed by addition of 100 nM Rotenone and 1μM Antimycin A (AmA) to determine spare respiratory capacity by OCR andthen 100 mM 2-DG to determine glycolytic reserve by ECAR.

For measuring catabolism (oxidation) of exogenous and endogenous FAs,cells activated in either Glu or Gal medium for 3 days were washed andtransferred to substrate-limited Glu or Gal media for overnightstimulation. Substrate limited media contained 0.5 mM Glu or Gal, 1 mMGlutaMAX, 0.5 mM carnitine (all form Sigma) and 1% dialyzed FBS. Sampleswere treated with either ETO or vehicle control 15 minutes before theassay. Palmitate: BSA or BSA was added just before the assay.

The contributions of fatty acid catabolism FAO to OCR was calculated asfollows: Basal respiration due to exogenous FAcatabolism/oxidation=(Basal Palm:BSA-ETO OCR rate−basal BSA-ETO OCRrate)−OCR due to uncoupling by FFA; uncoupling by FFA=after OMinjection, Palm:BSA-ETO OCR rate−BSA-ETO rate. Basal OCR due toendogenous FAs consumption=basal BSA-ETO OCR rate−basal BSA+ETO OCRrate.

Lipid and Glucose concentration measurement in tumor interstitial fluid.

Tumors interstitial fluid was collected as described (Wiig et al.,2003). Free FA species concentrations were determined by LC-MS. Absoluteconcentration of Glu was measured by LC-MS upon adding 13C6-Glu as theinternal standard.

Lentivector Transduction of CD8⁺T Cells

For in vitro experiments, 4×10⁶ enriched CD8⁺T cells were stimulated asdescribed above for 24-28 hours. Freshly concentrated lentivectors werespin-inoculated into activated CD8⁺T cells supplemented with polybrene(6 μg/ml, Santa Cruz) at 2000 rpm, 32° C. for 2 hours. Cells were washed20 hours after transduction, transferred to new CD3 antibody pre-coatedplates and stimulated for another 40 hours in medium supplemented withanti-CD28 and human IL-2 (100 U/ml, Roche) under normoxia or switched topart time hypoxia. Lentivector-transduced CD8⁺T cells were identified bysurface staining for Thy1.1 followed by analysis with a BD LSRII.

For in vivo adoptive transfer experiments, cells were washed 20 hoursafter lentivector transduction and cultured for an additional 48 hourswith medium supplemented with human IL-2 (100 U/ml) before celltransfer. Lentivector-transduced CD8+T cells were identified by surfacestaining for Thy1.1. Recipient Thy1.2+ mice at the time had beenchallenged with tumor cells 5 days earlier and had been vaccinated withAdC68-gDMelapoly 2 days earlier. For in vivo experiments CD8+T cellsfrom spleens of naïve C57Bl/6 mice were purified by negative selectionusing magnetic beads (MACS, STEMCELL Technologies). Enriched CD8+T cellswere stimulated with anti-CD3/CD28 antibodies for 24 hours prior tolentivector transduction via spin inoculation.

Isolation of Lymphocytes from Mice

PBMCs and splenocytes were harvested as described⁴⁰. Briefly, bloodsamples were collected by retro-orbital puncture and PBMCs were isolatedby Histopaque (Sigma) gradient. Spleens were harvested and single cellsuspension was generated by mincing with mesh screen in Leibovitz's L15medium and passing through 70 μm filter (Fisher Scientific, Waltham,Mass.). For both samples red blood cells were lysed by 1×RBC lysisbuffer (eBioscience, San Diego, Calif.). To obtain tumor-infiltratinglymphocytes, tumors were harvested, cut into small fragments and treatedwith 2 mg/ml collagenase P, 1 mg/ml DNase I (all from Roche) and 2% FBS(Tissue Culture Biologicals) in Hank's balanced salt solution (HBSS, 1×,Thermo Fisher Scientific) under agitation for 1 hour. Tumor fragmentswere homogenized, filtrated through 70 μm strainers and lymphocytes werepurified by Percoll-gradient centrifugation and washed with DMEMsupplemented with 10% FBS. Pre-experiments were conducted to ensure thatthis treatment did not affect any of the markers that were tested.

Antibodies, Staining and Flow Cytometry

Cells were stained with a PE-labeled Trp-1-specific MHC class I(H-2D^(b)) tetramer carrying the TAPDNLGYM peptide and anAlexa647-labeled HPV-16 E7-specific MHC class I (H-2D^(b)) tetramercarrying the RAHYNIVTTF peptide (obtained from the NIAID TetramerFacility). Lymphocytes were stained with anti-CD8-PerCPCy5.5 or-Alexa700, CD4-PercpCy5.5, CD44-PacBlue, LAG-3-APC or -PercpCy5.5,KLRGI-FITC, PD-1-PE-Cy7 or -Brilliant violet (BV) 605 (all fromBiolegend), 2B4-FITC (eBioscience) and Amcyan fluorescent reactive dye(Life Technologies).

For mitochondrial metabolic markers analysis, cells were stained withMitosox Red (5 μM, MROS) and DioC6 (40 nM, MMP) (Life Technologies) at37° C. for 30 minutes under either normoxia or hypoxia (for in vitrosamples cultured prior to staining under hypoxia). For fatty acid uptakeexperiments, cells stimulated under different conditions in vitro orisolated from spleen and tumors of mice bearing 2 weeks or 1 month-oldtumors were immediately incubated with 1 μM BODIPY FL C₁₆ (Life tech)for 30 mins at 37° C. Cells were washed twice with cold PBS beforesurface staining. For Cpt1a staining, cells were stained for surfacemarkers first followed by permeabilization with transcription factorbuffer set (BD Pharmingen, San Diego, Calif.). Cells were stained withanti-Cpt1a antibody or mouse IgG2b isotype control antibody (abcam) at 5μg/ml in 1× permwash for 45 mins at 4° C. For staining of T-bet, cellswere first stained for surface markers, then fixed and permeabilizedwith Foxp3/Transcription factor staining buffer and stained withT-bet-PE-Cy7 Eomes-FTIC (all from eBioscience) or primary antibodyagainst FoxO1 (C29H4, Cell Signaling Technology). Total FoxO1 wasfurther determined by anti-rabbit secondary antibody staining (CST). Forphorphorylated (p)Akt staining, cells were stained with BD Phosflowbuffer set and Phospho-Akt (CST) antibody.

For intracellular cytokine staining (ICS) of ex vivo lymphocytes ˜10⁶cells per samples were cultured in DMEM containing 2% FBS and Golgiplug(Fisher Scientific, 1.5 μl/ml) for 6 hours with either a peptide pool (5μg/ml for each peptide) including all CD8⁺T cell epitopes expressed bygD-Melapoly (mTrp-1₄₅₅₋₄₆₃: TAPDNLGYA, mTrp-1₄₈₁₋₄₈₉: IAVVAALLL,mTrp-2₅₂₂₋₅₂₉: YAEDYEEL, hTp-2₁₈₀₋₁₈₈: SVYDFFVWL, hTrp-2₃₄₃₋₃₅₇:STFSFRNAL, mTrp-2₃₆₃₋₃₇₁: SQVMNLHNL, hgp100₂₅₋₃₃: KVPRNQDWL,mBraf₅₉₄₋₆₀₂: FGLANEKSI) or the E7 peptide: RAHYNIVTTF (Genescript). Arabies virus glycoprotein peptide was used as a negative control.

For ICS performed with CD8⁺T cells stimulated in vitro, ˜10⁶ cells weretransferred to 96 well plates in the original medium and stimulated withPMA (500 ng/ml), ionomycin (20 μg/ml) and Golgiplug for 4 hours undereither normoxia or hypoxia. Staining was conducted as describedbefore.¹⁴ Cells were stained with antibodies to IFN-γ (APC or BV421),granzyme B (APC, Life Technologies) and perforin (PE, eBioscience).Cells were analyzed by an LSRII (BD Biosciences). Data were analyzedwith FlowJo (TreeStar).

BRDU Proliferation Assay

Mice were intraperitoneally injected with 1.5-2 mg/mouse of BrdUsolution and fed water-containing BrdU at a concentration of 0.8 mg/mlfor 24 hours before the assays. They were euthanized and lymphocytesamples were analyzed for BrdU incorporation. Cells were first stainedfor surface markers and then for intracellular BrdU (1:50 dilution)according to the manufacture's instruction (BD Bioscience).

HIF-1α and Glut1 Staining

For ex vivo assays mice were perfused immediately after euthanasia withPBS and heparin (10 units/ml) and then with 1 mM cobalt (II) chloride(Cocl₂, EMD Millipore) diluted in PBS. For both ex vivo and in vitroexperiments, lymphocytes isolation and staining before fixation wereperformed in medium containing 20004 Cocl₂. For staining, lymphocyteswere first blocked with 10% normal goat serum (Life Technology) for 30minutes at room temperature and then stained with anti-Glut1 primaryantibody or mouse IgG2a isotype control antibody (Abcam) at 1 μg/10⁶cells for 60 minutes at room temperature. Cells were washed and stainedwith PacBlue labeled-goat anti-mouse secondary antibody (1:2000dilution) together with antibodies to other cell surface markers for 30minutes. Cells were fixed, permeabilized, and stained for HIF-1α withanti-HIF-1α-Alexa700 antibody (R&D) using the FoxP3 buffer set(eBioscience).

Glucose Concentration Measurement in Tumor Interstitial Fluid

Mice bearing week 2 or 1 month-old tumors were euthanized. Tumors wereremoved and interstitial fluid was collected as described³⁷. Gluconcentration was determined with Glu meters.

Cell Culture and Isotopic Labeling.

For ¹³C₆-Glu/Gal tracing in vitro, cells were cultured from the onset ofthe assays in Glu-free RPMI medium with 10 mM Glu/Gal-¹³C₆ (Sigma) for 4days. For ¹³C₁₆-palmitate tracing in vitro, cells were stimulated for 3days in Glu or Gal medium. On the night of day 3, some samples weretransferred to 1% O₂ for overnight culture. ¹³C₁₆-palmitate (Sigma) wasfirst dissolved in 100% ethanol at 200 mM and conjugate to fattyacid-free BSA (Sigma) at a 5:1 molar ratio to a final concentration of 8mM-¹³C₁₆-palmitate-BSA by vortexing at 37° C. for 3-4 hours withsonication. On day 4, samples were pelleted and replated in fresh mediumwith 10% delipidated FBS (Cocalico Biologicals, Reamstown, Pa.) and 400μM ¹³C₁₆-palmitate-BSA. Hypoxia samples were returned to 1% O₂. Allsamples were cultured for another 4 hour. Dead cells were removed byMACS. Samples were pelleted at 4000 rpm for 5 minutes. All collectionprocedures were conducted at 4° C.

Cell numbers in each sample were determined. Metabolism was quenched andmetabolites were extracted by adding 1 ml-80° C. 80:20 methanol:waterper million cells. After 20 min of incubation on dry ice, samples werecentrifuged at 10000 g for 5 min. Insoluble pellets were re-extractedwith 1 ml-80° C. 80:20 methanol:water on dry ice. The supernatants fromtwo rounds of extraction were combined, dried under N₂, resuspended in 1ml water per million cells. Metabolites were normalized to cell number.

For ¹³C₆-Glu tracing in vivo, tumor-bearing mice were fasted for 16hours overnight. ¹³C₆-Glu was diluted in PBS and given i.p. to mice at 2g/kg. Spleen and tumors were collected 30 minutes after injection. ForU¹³C₁₆-palmitate tracing mice were fasted for 16 hours, they were then¹³C₁₆-palmitate dissolved in polyethylene glycol 300 (Hampton Research,Aliso Viejo, Calif.) at 0.5 g/kg by oral gavage. 1-hour later¹³C₁₆-palmitate dissolved in intralipid, 20% (Sigma) was given i.v. 150mg/kg. Mice were euthanized 1 hour after the i.v. injection and spleensand tumors were collected. Tissues were processed and cells wereisolated on ice as rapidly as possible. CD8⁺CD44⁺T cells from pooledspleens and tumors were stained and sorted at 4° C., cell numbers weredetermined. Metabolites were extracted with −80° C. 80:20methanol:water, dried under N₂, resuspended in 1 ml water. Metaboliteswere normalized to cell number. Contribution of ¹³C to TCA cyclemetabolites was calculated as [(m+1)*1+ . . . (m+n)*n]/{[(m+0)+ . . .+(m+n)]*n}*100%, where m+0 is the normalized signal intensity of ametabolite in ¹²C form, m+n indicates normalized signal intensity foreach form of ¹³C labeled metabolite, n indicates the total number ofcarbon atoms in that metabolite.

LC-MS Instrumentation and Method Development.

Glycolytic and TCA metabolites were analyzed by reversed-phaseion-pairing chromatography coupled with negative-modeelectrospray-ionization high-resolution MS on a stand-alone orbitrap(Thermo) (Lu et al., 2010). Carnitine species were analyzed byreversed-phase ion pairing chromatography coupled with positive-modeelectrospray-ionization on a Q Exactive hybrid quadrupole-orbitrap massspectrometer (Thermo); Liquid chromatography separation was achieved ona Poroshell 120 Bonus-RP column (2.1 mm×150 mm, 2.7 μm particle size,Agilent). The total run time is 25 min, with a flow rate of 50 μl/minfrom 0 min to 12 min and 200 μl/min from 12 min to 25 min. Solvent A is98:2 water:acetonitrile with 10 mM amino acetate and 0.1% acetic acid;solvent B is acetonitrile. The gradient is 0-70% B in 12 min. Allisotope labeling patterns were corrected for natural ¹³C-abundance.

Gene Expression Analysis

Lymphocytes were isolated from spleens and tumors of mice (tumor-bearingor normal) at different time points and stained with dyes and antibodiesto live cells, CD8⁺, CD44⁺ and the Trp-1 and E7 tetramers. Forco-adoptive transfer experiments, CD8⁺CD44⁺ T donor cells of differentorigin were recovered from spleen and tumors of recipient mice threeweeks later by antibodies staining and sorting. Cells were sorted (MonoAstrios, Beckman Coulter) on ice into Trp-1 or E7 tet⁺CD44⁺CD8⁺T cellsinto RNAprotect cell reagents (QIAGEN).

For in vitro cultured samples ˜10⁶ cells/sample were processed on ice toremove dead cells using manual cell separation columns and Mini/MidiMACS separators (Miltenyi Biotec). For lentivector transduction assays,transduced cells were further purified based on Thy1.1 expression usingMACS. RNA was isolated from purified cells using RNeasy Mini kits(Qiagen) and RNA concentrations were determined using Nanodrop (ThermoScientific). cDNAs were obtained by reverse transcription using the highcapacity cDNA reverse transcription kit (Life Technologies). Relativequantitative real-time PCR analyses were performed using 7500 FastReal-Time PCR system (Life Technologies). b-2 microglobulin or GAPDHwere used as internal controls. GAPDH or 13-2 microglobulin were used asinternal controls. Table 1 shows the metabolic pathways, gene name andforward and reverse primers for the gene expression analysis describedin Example 1. Vector NTI was used for primers design.

TABLE 1 1 Gene name (Forward-F SEQ Metabolic and  ID Pathways Reverse-RPrimer Sequences NO Glucose mGLUT1-F TGTGGGAGGAGCAGTGCTCG 2 metabolismmGLUT1-R TGGGCTCTCCGTAGCGGTG 3 mHK2-F TGATCGCCTGCTTATTCACGG 4 mHK2-RACCGCCTAGA AATCTCCAGA  5 AGG mPGK1-F ATGTCGCTTTCCAACAAGCT G 6 mPGK1-RTGGCTCCATTGTCCAAGCAG 7 mIDH3a-F TGGGTGTCCAAGGTCTCTCG 8 mIDH3a-RTCTGGGCCAATTCCATCTCC 9 mMDH2-F TTGGGCAACCCCTTTCACTC 10 mMDH2-RTGTGACTCAGATCTGCTGCCAC 11 Lipid mPPARa-F AGCCCCATCTGTCCTCTCTCC 12metabolism mPPARa-R TCCAGAGCTCTCCTCACCGATG 13 regulation, mSLC27A4-FTGAGTTTGTGGGTCTGTGGCTAGG 14 FAs uptake, mSLC27A4-R AAGACAGTGGCGCAGGGCATC15 TG synthesis mSLC27A2-F TGCTGCTGCTGCCTCTGCTG 16 and  mSLC27A2-RAGGATGGTACGCACGGGTCG 17 lipolysis mDGAT1-F ACCTGGCCACAATCATCTGCTTC 18and FA mDGAT1-R TTGGCCTTGACCCTTCGCTG 19 synthesis mDGAT2-FAGCATCCTCTCAGCCCTCCAAG 20 mDGAT2-R TAGCACCAGGAAGGATAGGACC 21 mPNPLA2-FTTCCCGAGGGAGACCAAGTG 22 mPNPLA2-R TGCCGAGGCTCCGTAGATG 23 mLIPA-FTGCTTTCTCGGGTGCCCAC 24 mLIPA-R TCCTCACCAGGATATCCCCAG 25 PeroxisomalmACAA1α-F TCCGCTAGGTTCCCGCAGG 26 FAO mACAA1α-R ACAGAAGCTCGTCGGGGGTG 27mEHHADH-F AAAGTTCGEAAAGGGCAAGG 28 mEHHADH-R TCGCCCAGCTTCACAGAGC 29mACOX1-F TCCCGATCTGCGCAAGGAG 30 mACOX1-R TGTTCTCCGGACTACCATCCAAG 31mHSD17B4-F TTGTGAACGACITAGGAGGGGAC 32 mHSD17B4-R AAATGTGTCCAGTGCCGTCGGC33 Mito- mACADVL-F ACC CTCTCCTCTGATGCTTCCAC 34 chondrial mACADVL-RTGAGCACAGATGGGTATGGGAAC 35 FAO mACADM-F AAGCAGGAGCCCGGATTAGG 36 mACADM-RTCCCCGCTTTTGTCATATTCC 37 Ketone Body mBDH1-F TCGCCATACTGCATCACCAAG 38Metabolism mBDH1-R TGCCAGGTTCCACCACACTG 39 ROS mNOX1-FAGAAATTCTTGGGACTGCCTTGG 40 production/ mNOX1-R TGCCCCTCAAGAAGGACAGC 41detoxi- mSOD1-F ACAGGATTAACTGAAGGCCAGC 42 fication mSOD1-RTTGCCCAGGTCTCCAACATG 43 and Electron mCAT-F TGACATGGTCTGGGACTTCTGG 44transport mCAT-R AGCCATTCATGTGCCGGTG 45 chain (ETC) mCOX5B-FACCCGCTCCATGGCTTCTG 46 mCOX5B-R AGTCCCTTCTGTGCTGCTATCATG 47

Differences in transcript expression levels are visualized in heatmaps.Values were log transformed to show ratios of differences. Color scalewas set as −2 (lower expression, deep blue) to 2 (higher expression,deep red).

Isotope Labeling In Vivo

Tumor-bearing mice were fasted for 16 hours. 13C6-Glu (Cambridge)diluted in PBS was given i.p. to mice at 2 g/kg. Spleens and tumors werecollected 30 minutes later. 13C16-potassium palmitate was conjugated toFAfree BSA (6:1 molar ratio) and given to mice at ˜0.35 g/kg by oralgavage. 1-hour later 13C16-palmitate-BSA was given i.v. at 125 mg/kg.Spleens and tumors were collected 30 mins later and cells were isolatedon ice. Tumor samples were weighed and flash frozen in liquid nitrogen.CD8+CD44+T cells from pooled samples were stained and sorted at 4° C.Metabolites were extracted with −80° C. 80:20 methanol:water, driedunder N2 and resuspended in water at 100 mg tissue/ml or 106 cells/100μl.

Statistical Analysis

Significance of differences between 2 populations was calculated bystudent's t test; significance of differences among multiple populationswas calculated by one-way or two-way ANOVA using GraphPad Prism 6.Differences in survival were calculated by Log-rank Mantel-Cox test.Significance was set at p-values of or below 0.05. Type I errors werecorrected for multiple comparisons using the Holm-Sidak method.

Example 2: CD8+T Cells Become Functionally Impaired within the TMEIndependent of Recognition of their Cognate Antigen

To test if the fate of CD8+TILs depends on continued antigenrecognition, we used two vaccines in a transplantable mouse melanomamodel. One, termed AdC68gD-Melapoly⁴⁰ is an adenovirus (Ad)-basedpolyepitope vaccine that elicits MAA-specific CD8+T cell responses,mainly to the Trp-1₄₅₅₋₄₆₃ epitope; the other, termed AdC68-gDE7¹⁵,stimulates CD8+T cells to E7.

We vaccinated mice bearing 3-day old B16BrafV600E tumors and normal micewith AdC68-gDMelapoly mixed with AdC68-gDE7. Vaccination delays tumorprogression (FIG. 1A). Experiments indicating the numbers of tetramer(tet)+ Trp-1- and E7-specific CD8+T cells/10⁶ live cells in spleens(Spl) and tumors of mice that had or had not been challenged with tumorcells 3 days (d) before vaccination (n=7-10/group), revealed that bothTrp-1 and E7-specific CD8+T cells accumulate within tumors where theycontract more rapidly in TME than in periphery during tumor progression(data not shown).

The % of bromodeoxyuridine (BrdU) incorporation into Trp-1- andE7-specific specific CD8+T cells from spleens and tumors of mice thathad been challenged with B16 cells 3 days before vaccination (n=5/timepoint) was measured. BrdU was administered one day before euthanasia for24 hours on days 9, 19 or 29 after vaccination. Measurements were takenon day 10, day 20 and month 1 (n=5, representative of 2 experiments).Only Trp-1 specific CD8+ T cells proliferate in the tumor. Numbers ofTrp-1-specific CD8+TILs declined by more than 90% between 10 days and 1months after vaccination while E7-specific CD8+TILs declined less by˜80%. This prominent reduction of Trp-1-specific CD8+TIL numbers wasobserved even though they proliferate within tumors. Nevertheless theirproliferation decreases over time despite continued presence of Trp-1antigen (not shown).

Mean fluorescent intensity (MFI) and representative histograms for PD-1and LAG-3 on specific CD8+T cells were produced from spleens and tumorsat 2 weeks (wk) or 1 month (mo) after vaccination. Both Trp-1-specificand bystander E7-specific CD8+ T cells from 2-week and, to a morepronounced extent, 1-month tumors increase expression of exhaustionmarkers PD-1 and LAG-3 within the TME (data not shown). BothTrp-1-specific and bystander E7-specific CD8+ T cells lose functionswithin the TME.

This, combined with declining frequencies of antigen-specific CD8+TILsproducing effector molecules, such as lytic enzymes or interferon(IFN)-γ and reduced polyfunctionality (FIG. 1B), suggests thatvaccine-induced TILs regardless of their epitope specificity,differentiate towards ‘exhaustion’ during tumor progression. Ad vectorsused for vaccination persist at low levels thus maintaining highfrequencies of fully functional effector CD8+ T cells (Tatsis et al.,2007).

In another experiment, graphs were generated showing MFI of CD62L,CD127, KLRG1 and Eomes on/in specific CD8+ T cells from spleens andtumors with representative histograms for Trp-1-specific CD8+T cells andCD44-naive T cells (FIG. 1C). Levels of differentiation markers CD62L,CD127, KLRG1 and Eomes on/in vaccine-induced CD8+ T cells from spleensand tumors remained comparable. Thus, factors other than chronic antigenexposure contribute to CD8+TIL exhaustion.

Metabolic stress dictates cellular fate^(26,16,23) and potentiallyaffects functions and survival of CD8+TILs independent of their antigenspecificity. During tumor progressionTrp-1- and E7-specific CD8+TILsgradually lose mitochondrial membrane potential (MMP) (FIG. 2A), whichis essential for ATP production and increase levels of toxicmitochondrial reactive oxygen species (MROS). MMP^(lo)MROS^(hi) Trp-1-and to a lesser extent E7-specific CD8+TILs become prevalent in latestage tumors (FIG. 2B), while corresponding CD8+T cells from spleensremain largely MMP^(hi)MROS^(lo). These data suggest that CD8+TILsincreasingly experience metabolic stress within growing tumors.

As shown herein, continued stimulation seems to accelerate theimpairment.

Example 3: Hypoxia Through HIF-1a Increases Lag-3 Expression and ReducesT Cell Functions

Trp-1- and E7-specific CD8+TILs are progressively subjected to hypoxiaduring tumor progression as shown by their enhanced expressions ofHIF-1α, a transcription factor that stabilizes under hypoxia, and itsdownstream target Glut1, which facilitates Glu uptake, in/onvaccine-induced CD8+TILs from 1-month (FIG. 2C), but not small 2-weektumors (data not shown).

To determine how hypoxia affects CD8+T cells, we stimulated enriched,activated CD8⁺ T cells in vitro continuously for 96 hours under normoxia(21% O₂) or for the last 16 hours under hypoxia (1% O₂) Hypoxia reducesblast formation (FIG. 8A) and increases expression of HIF-1α and Glut1(FIG. 8B). The O₂ consumption rate (OCR), a measure of OXPHOS, decreasesunder hypoxia while the extracellular acidification rate (ECAR), ameasure of glycolysis, increases (FIG. 8C). The T cells' MMP decreasesand MROS increases, leading to a rise in the proportion of MMPloMROShiCD8+T cells (data not shown) reminiscent of the metabolic profile ofvaccine-induced TILs in late-stage tumors. Hypoxia reduces PD-1 butaugments LAG-3 expression (FIG. 8D), suggesting that PD-1 declines andLAG-3 increases upon enhanced glycolysis driven by HIF-1α signaling.CD8+T cells cultured under hypoxia reduce T-bet expression (FIG. 8D),decrease production of effector molecules and lose polyfunctionality(FIG. 8E), indicating that hypoxia impairs CD8+T cell functions.

An alternative previously described protocol (Doedens et al., 2013) thattests the effect of hypoxia on resting CD8+T cells was conducted bystimulating T cells for 48 hours. The CD8+T cells upon activation areswitched to IL-2 containing medium without antibodies to CD3 or CD28stimulation and maintained at a relatively resting stage in IL-2 priorto hypoxia. The effect of hypoxia was assessed by subjecting cellscultured in IL-2 to 1% O₂ for the last 36 hours. FIG. 8F shows blastformation; normalized % of live IL-2 maintained CD8+T cells formingblasts under hypoxia (white) compared to those cultured under normoxia(dark gray) using this protocol. This protocol, has no effect on blastformation (FIG. 8F) or PD-1, although LAG-3 increases (FIG. 8G) andT-bet decrease (FIG. 8G). Granzyme B (GzmB) production increases whileproduction of other effector molecules and polyfunctionality decline(FIG. 8H). As vaccine-induced CD8+T cells are unlikely to rest beforeinfiltrating hypoxic areas of a TME, we used the protocol of continuousCD8+T cell activation for subsequent hypoxia experiments.

Solid tumors commonly lack O₂. The data show that Trp-1- and E7-specificCD8+TILs within the TME increasingly experience hypoxia during tumorprogression as shown by enhanced expressions of HIF-1α, a transcriptionfactor that stabilizes under hypoxia, and its downstream target Glut1,which facilitates Glu uptake, in/on vaccine-induced CD8+TILs fromlate-stage (FIG. 2C) but not small week 2 tumors (data not shown).

To test the effect of hypoxia, we stimulated CD8+T cells in vitro for 4days in regular Glu-rich medium under normoxia (21% O₂) or subjectedthem to hypoxia (1% O₂) for the last 16 hours of culture. Hypoxiaaffects T cell stimulation as evidenced by reduced blast formation (FIG.8A). CD8+T cells activated under hypoxia increase expression of HIF-1αand Glut1 (FIG. 8B). They become metabolically stressed as evidenced bydecreases in MMP and rises in MROS, leading to an increase in theproportion of MMPloMROShi CD8+T cells (FIG. 8C) reminiscent of themitochondrial metabolic profile of vaccine-induced TILs in late-stagetumors. Hypoxia reduces PD-1 but augments LAG-3 expression (FIG. 8D),suggesting that PD-1 declines and LAG-3 increases under conditions thatpromote glycolysis such as intensified HIF-1α signaling. CD8+T cellscultured under hypoxia reduce T-bet expression (FIG. 8D), decreaseproduction of effector molecules and lose polyfunctionality (FIG. 8E).

A different previously described protocol¹⁰, in which CD8+T cells uponthe initial activation are rested in IL-2 prior to hypoxia, has noeffect on blast formation (FIG. 8F) or PD-1 levels, although LAG-3expression increases (FIG. 8G) and T-bet levels decrease (FIG. 8G).Granzyme B (GzmB) production increases while production of othereffector molecules and polyfunctionality decline (FIG. 8H). Asvaccine-induced CD8+T cells are unlikely to rest before infiltratingtumors, we used the protocol of continuous CD8+T cell activation forsubsequent hypoxia experiments.

HIF-1α correlates with expression of LAG-3 on TILs or CD8+T cellssubjected to hypoxia. To determine whether HIF-1α directly promotesLAG-3 expression and whether this affects CD8+T cell functions, weknocked down HIF-1α transcripts by transducing CD8+T cells withlentivectors that express either short-hairpin (sh)RNA to silence HIF-1αor a control sequence, together with a Thy1.1 selection marker (data notshown). HIF-1α silencing/knockdown reduces expression of HIF-1α in CD8+Tcells stimulated in vitro under hypoxia (See FIG. 3A), concomitantlydecreases LAG-3 but not PD-1 (see FIG. 3A) and improves production ofGzmB and IFN-γ (see FIG. 3B). HIF-1α positively correlates withexpression of LAG-3 on TILs or CD8+T cells subjected to hypoxia.

To study whether HIF-1α contributes to the CD8+TILs' co-inhibitorexpression and loss of functions, we activated enriched CD8+T cells invitro. The enriched CD8⁺T cells were transduced with lentivectors (i.e.,lentivirus-based vectors) expressing control or HIF-1α targeting shRNAand Thy1.1. The viruses were transferred i.v. into recipient mice whichhad been challenged with tumor cells 5 days earlier and been vaccinatedwith AdC68-gDMelapoly 2 days earlier (i.e., Thy1.2⁺ tumor-bearing,AdC68-gDMelapoly-vaccinated mice).

Trp-1-specific Thy1.1⁺CD8⁺T cells were recovered from tumors about 2 to3 weeks after transfer. We analyzed the transferred T cells ˜2-3 weekslater using samples from mice bearing similar sized tumors. Cells fromtumors were first gated on mononuclear cells, singlets, live cells andCD8⁺T cells. They were further gated on Thy1.1⁺ cells andTrp-1-tetramer⁺CD44⁺ cells.

Knocking down HIF-1α (FIG. 3A) reduces the Trp-1-specific CD8+TILs'expression of LAG-3 without affecting PD-1 (FIG. 3A) and significantlyimproves the frequencies and effector functions of MAA-specific CD8+TILs(FIG. 3B). The T cells' frequencies and functions (FIG. 3C). Productionof perforin increased upon HIF-1α-silencing in vivo but not in vitro,which may reflect that other conditions such as differences in supply ofnutrients contribute to the effect of hypoxia on activated CD8+T cells.These data support that hypoxia through increased HIF-1α signalingdirectly enhances LAG-3 expression and dampens effector functions ofCD8+TILs.

Collectively the data show that hypoxia through HIF-1α leads tofunctional impairment of activated CD8+T cells. Hypoxia-induced HIF-1αdirectly drives co-inhibitor LAG-3 expression and impairs effectorfunctions of activated CD8+T cells in vitro. HIF-1α increases glucoseuptake and enzymes of glycolysis. It reduces energy production throughthe TCA cycle by activating PDK1 which inactivates pyruvatedehydrogenase. Within a glucose poor environment increased HIF-1αsignaling is counterproductive for TAA-specific CD8+ T cells. The datafurther suggest that a HIF-1α-driven metabolic switch to glycolysis iscounterproductive for T cell functions within an O₂ and Glu-depletedTME.

Example 4: Activated CD8+T Cells Lacking Both Glu and O₂ Enhance FACatabolism

Not only O₂ but also Glu declines within the TME during tumorprogression presumably due to its consumption by tumor cells. Thecollective effects of hypoglycemia and hypoxiaon activated CD8+T cellswere studied by stimulating them in vitro in Glu medium with2-deoxy-D-glucose (2-DG), a glycolysis inhibitor. Alternatively, Glu wasreplaced by galactose (Gal). Cells were cultured under normoxia orshort-term hypoxia. The extracellular acidification rate (ECAR), ameasure of glycolysis, declines in T cells cultured with 2-DG or Gal.

The O₂ consumption rate (OCR), a measure of OXPHOS, decreases in T cellscultured with 2-DG but increases in the presence of Gal (FIG. 9A).Either condition augments the OCR/ECAR ratio, suggesting increasedenergy production through OXPHOS Cells cultured with 2-DG or Galcompared to those grown with Glu express more PD-1, suggesting that theuse of OXPHOS is linked to high PD-1 expression on activated CD8+ Tcells (FIG. 9B-9C). Compared to cells cultured with Glu, those grownwith limited access to Glu under both normoxia and hypoxia decreaseT-bet expression (FIG. 9D) and lose functions (FIG. 9E). Poly-functionsof CD8+T cells without access to Glu are better preserved if cells arealso subjected to hypoxia (FIG. 9D). These data suggest that underhypoxia cells with limited supply of Glu may more adjust theirmetabolism to support their functions.

To study the metabolic pathways used by CD8+T cells with limited Glu andO₂ supply, we measured transcripts for factors that participate innutrient consumption and energy production by quantitative Real-Time(qRT) PCR (FIG. 15F, FIG. 4B and Table 2).

TABLE 2 Function Name Abbreviaton Glucose uptake glucose transporter 1Glut1 Glycolysis hexokinase 2 Hk-2 phosphoglycerate kinase Pgk1 TCAcycle isocitrate dehydrogenase subunit alpha Idh3a monodehydroascorbatereductase 2 Mdh2 Regulation of FA peroxisome proliferator-activatedreceptor Ppar-α melabolism alpha FA transport solute carrier family 27a4Slc27a4 solute carrier family 27a2 Slc27a2 Triglyceride (TG)Diacylglycerol-O-acyltransferase 1 Dgat1 synthesisDiacylglycerol-O-acyltransferase 2 Dgat2 Triglyceride (TG) Lipase A Lipacatabolism patatin-like phospholipase domain Pnpla2 containing 2Peroxisomal FAO acetyl-CoA acyltransferase 1 Acaa1a CoA dehydrogenaseEhhadh acyl-CoA oxidase 1 Acox1 hydroxysteroid (17-beta) dehydrogenase 4Hsd17b4 Mitochondrial FAO carnitine palmitoyltransferase 1a Cpt-1aacyl-CoA dehydrogenase, very long chain Acadvl acyl-CoA dehydrogenase,C4 to C12 Acadm straight chain Metabolism of ketone 3-hydroxybutyratedehydrogenase 1 Bdh1 bodies ROS metabolism NADPH oxidase 1 Nox1superoxide dismutase 1 Sod1 catalase Cat Electron transport chaincytochrome c oxidase subunit Vb Cox5b

Upon short-term hypoxia CD8+T cells stimulated with limited access toGlu compared to those stimulated in Glu-rich medium decrease transcriptsfor enzymes of glycolysis, the tricarboxylic acid (TCA) cycle, ROSdetoxification, and the electron transport chain (ETC), but increasetranscripts for receptors and enzymes of FA uptake, triglyceride TGturnover, peroxisomal and mitochondrial FA catabolism.

This pattern is closely mirrored by Trp-1- and E7-specific CD8+TILs from1-month tumors compared to those from small 2 week-old tumors,indicating that metabolically stressed CD8+T cells increasingly rely onFA catabolism. Changes in transcripts during tumor progression are notdriven by differentiation of TILs towards a more resting stage, as theyare distinct from differences in vaccine-induced splenic CD8+T cellstested at 3 months vs 2 weeks after vaccination.

To directly measure effects of Glu and O₂ deprivation on CD8+T cellmetabolism, we analyzed the intensity of metabolites by lipidchromatography-mass spectrometry (LC-MS). Metabolites involved in FAmitochondrial transport and catabolism, i.e., acetylcarnitine,palmitoylcarnitine and the ketone body 3-hydroxybutyrate, increase incells stimulated in vitro in Gal medium and this is further enhancedunder hypoxia (FIG. 10A). ¹³C₆-Glu/Gal or ¹³C₁₆-palmitate isotopetracing show that CD8+T cells activated in Glu medium and short-termhypoxia or in medium with limited access to Glu under normoxia orhypoxia compared to those activated in Glu medium under normoxia havereduced carbohydrate-derived TCA cycle metabolites (FIG. 10B).¹³C₁₆-palmitate-derived carbons are increasingly incorporated intoacetyl-CoA and TCA cycle metabolites (FIG. 10C). Moreover, cellsstimulated under hypoxia show higher percentages of¹³C₁₆-palmitate-derived acetyl-CoA than those cultured in the samemedium under normoxia, suggesting that hypoxia further increases fattyacid catabolism. This is also supported by increased FA uptake (FIG.10D) and enhanced oxidation of endogenous and exogenous FAs (FIG. 10E)by CD8+T cells activated with limited access to Glu and/or O₂. Thesedata indicate that activated CD8+T cells deprived of Glu increasinglyrely on FA catabolism to produce energy through OXPHOS. Surprisinglyalthough OXPHOS requires O₂, hypoxia increases this metabolic switch.

We next studied the metabolism of activated CD8+T cells directly in vivoby stable isotope tracing. Mice bearing 3-day tumors were vaccinatedwith AdC68-gDMelapoly and AdC68-gDE7. Two weeks or one month later micewere given ¹³C₆-Glu i.p. 30 minutes later, TILS are isolated, andCD8+CD44+ cells are isolated and their metabolites analyzed. Levels ofglycolysis metabolites and ¹³C incorporation into TCA cycleintermediates were analyzed in CD44+CD8+T cells from spleen and tumors.The intensity of glycolysis intermediates glucose-6-phosphate (G6P) and3-phosphoglycerate (3PG) in TILs declines during tumor progression (FIG.4C), indicating reduced glycolysis. The contribution of Glu-derivedcarbon to TCA cycle intermediates declines comparing CD44+CD8+T cellsfrom late to early stage tumors or from tumors to spleens (FIG. 4D),confirming that TILs decrease Glu catabolism.

We further performed ¹³C₁₆-palmitate tracing in mice bearing 2-week or1-month tumors. Mice were administered ¹³C₁₆-palmitate-BSA per os/i.v.Sixty minutes after feeding and 30 minutes after injection, the TILSwere isolated and the CD8+CD44+ cells were isolated and metabolitesanalyzed. The intensities of acylcarnitine species, the ketone bodies3-hydroxylbutyrate and acetoacetate increase in TILs during tumorprogression (FIG. 4E). Moreover, the contribution of¹³C₁₆-palmitate-derived ¹³C to TCA metabolites becomes higher inCD44+CD8+T cells from 1-month tumors compared to those from 2-weektumors or 1-month spleens (FIG. 4F), supporting the TILs' enhancedreliance on FA catabolism during tumor progression. In splenicCD44+CD8+T cells tested at different time points after vaccination,¹³C₁₆-palmitate-derived carbon incorporation into TCA cycle metabolitesremains stable or decreases over time. CD62L and CD127 expression aremarkedly lower on CD44+CD8+TILs from 1-month compared to those from2-week tumors (data not shown), confirming the enhanced FA catabolism bylate-stage CD8+TILs is not reflective of their differentiation towardsmemory.

The metabolic switch of TILs towards FA catabolism is facilitated byhigh abundance of different free FA species within the melanomainterstitial fluid (FIG. 4G), enhanced uptake of FAs (FIG. 4H) andincreased expression of the FA oxidation (FAO) rate-limiting enzymeCpt1a (FIG. 4I) in vaccine-induced CD8+T cells from late stage tumors.

Trp-1- and E7-specific TILs from late-stage tumors increase FA uptake(FIG. 4H) and expression of the fatty acid catabolism rate-limitingenzyme Cpt1a (FIG. 4I) compared to TILs from early tumors orcorresponding T cells from spleens suggesting their increased relianceon FA catabolism. Both FA uptake and Cpt1a levels remain low andcomparable in CD8+T splenocytes induced by vaccination 2 weeks or 1month earlier, suggesting metabolic changes observed in TILs are not dueto their differentiation towards a more resting ‘memory’ stage.Comparisons of FA metabolites show higher levels of acyl-carnitinespecies and the ketone body acetoacetate in CD44+CD8+TILs formlate-compared to early-stage tumors (FIG. 4E). ¹³C contribution from¹³C₁₆-palmitate to TCA metabolites increases in CD44+CD8+TILs duringtumor progression (FIG. 4G), further supporting their enhanced relianceon fatty acid catabolism. In splenic CD44+CD8+T cells tested atdifferent times after vaccination, ¹³C₁₆-palmitate derived ¹³Cincorporation into citrate or malate remains stable or decreases overtime (FIG. 4F).

This data show that vaccine-induced CD8+ TILs in late-stage tumorsenhance co-inhibitors PD-1 and LAG-3 expression, lose effector functionsand polyfunctionality, experience enhanced metabolic stress and decreaseglucose metabolism and increasingly rely on FA catabolism.

Overall these data strongly indicate that CD44+CD8+T cells deprived ofGlu increasingly rely on FA catabolism to produce energy through OXPHOS.Surprisingly this further increases upon hypoxia, although OXPHOSrequires O₂.

Example 5: Enhanced Reliance on FA Catabolism Increases Pd-1 Expressionand is Essential to Maintain CD8+T Cell Functions Under MetabolicallyStressed Condition

To further assess the impact of FA catabolism on CD8+T celldifferentiation, we stimulated CD8+T cells in presence of fenofibrate(FF), an agonist of PPARα that enhances FA catabolism, or etomoxir(Eto), an irreversible inhibitor of Cpt1 that decreases mitochondrialfatty acid catabolism (FIG. 6A). In vitro FF-treated cells stimulated inGlu or Gal medium compared to diluent-treated cells increase fatty acidcatabolism as shown by their enhanced transcripts of factors involved inFA catabolism (data not shown) and increased FA uptake (FIG. 12A). CD8+Tcells stimulated in either Glu or Gal media decrease OCR in presence ofEto (FIG. 12B), confirming the drug's inhibitory effect on fatty acidcatabolism. OCR declines more in cells cultured with Gal and Eto, andfurther decreases when cells are also subjected to hypoxia, againconfirming the cells' increased reliance on fatty acid catabolism whenGlu and O₂ are limited. Under hypoxia PD-1 increases with addition of FFbut decreases in presence of Eto (FIG. 12C). FF increases while Etodecreases functions and polyfunctionality of CD8+T cell cultured withlimited access to Glu and O2. These results show that FA catabolismpromotes PD-1 expression and effector functions of metabolicallystressed CD8+T cells.

As shown in FIG. 4B, T cells experiencing metabolic stress increasetranscripts of enzymes participating in TG turnover. To assess if CD8+Tcells under such conditions mobilize TGs to fuel fatty acid catabolismand OXPHOS, we added Orlistat (OS), an inhibitor of the lipolysis enzymelipa, or Amidepsine A (AmA), an inhibitor of the TG synthesis enzymesDgat1 and Dgat2, to CD8+T cells stimulated under different conditions(FIG. 6A). Basal OCR decreases in CD8+T cells cultured with either drugin Glu or Gal media (data not shown) under short-term hypoxia,suggesting that TG turnover provides fuels for OXPHOS. Under hypoxiaPD-1 decreases with addition of OS regardless of other cultureconditions and with addition of AmA to Gal medium (FIG. 12C). AmA and toa lesser degree OS decrease functions of T cells cultured in Gal mediaand subjected to hypoxia. These data indicate that T cells activatedunder hypoxia use substrates from TG turnover for OXPHOS as OCRdecreases in presence of AmA or OS. However TGs are not essential foreffector functions unless CD8+T cells are concomitantly subjected tohypoglycemia.

To assess how increased FA catabolism affects CD8+TIL functions, wevaccinated mice expressing CD90.2 and congenic for CD45, and treatedthem for 3 weeks daily with FF (CD45.1 mice) or diluent (CD45.2 mice).Splenocytes from these groups were mixed such that Trp-1-specific CD8+Tcells were present at equal numbers. The mixture was transferred intoCD90.1+ recipient mice, which had been challenged with tumor cells andvaccinated 3 days later. Cells were transferred 2 days after vaccination(FIG. 5B). Immediately before transfer FF and diluent-treated Trp-1- andE7-specific CD8+T cells from donor mice have similar functions withcomparable surface expression of CD127, indicating that FF does notaffect T cell activation or memory formation. FF-treated CD8+T cellsbefore transfer enhance OCR, which is blocked by Eto, indicating that FFconditions vaccine-induced CD8+T cells to enhance fatty acid catabolism.Donor-derived vaccine-induced CD8+TILs were analyzed 3 weeks aftertransfer (data no shown). Compared to control-treated donor TILs,FF-treated donor-derived CD44+CD8+TILs show enhanced levels oftranscripts for factors involved in lipid metabolism and increased PD-1expression that reaches significance for Trp-1 specific cells. Inaddition, frequencies and functions of Trp-1- and E7-specific CD8+TILsoriginating from FF-treated donor exceed those from control donors. Whentransferring splenocytes of FF- or diluent-treated mice into separatecohorts of tumor-bearing mice, the former significantly delay tumorgrowth (FIG. 6G). Collectively these data confirm that enhanced FAcatabolism increases PD-1 expression on TA-specific TILs, whilepreserving their functions.

To further study whether FA catabolism maintains functions ofmetabolically stressed CD8+T cells, we stimulated CD8+T cells from PPARαKO mice in vitro and compared them to those from wildtype (wt) mice.Transcripts for most factors involved in the TCA cycle and lipidmetabolism are higher in PPARα KO compared to wt CD8+T cells whenstimulated in Glu medium and under hypoxia. This profile reverses incells cultured in Gal medium and low O₂, suggesting that inactivation ofPPARα significantly decreases FA catabolism of CD8+T cells culturedwithout Glu (data not shown). PPARα KO compared to wt CD8+T cellsexpress lower levels of PD-1 when cultured with Gal-medium regardless ofO₂ levels (FIG. 13A). Their functions are lower compared to those of wtCD8+T cells cultured under the same conditions (FIG. 13B), supportingthat FA catabolism is required to maintain effector functions of CD8+Twith limited access to Glu.

To further explore the effect of FA catabolism on vaccine-induced TILs,we used an adoptive transfer system, in which splenocytes from PPARα KOand wt CD45 congenic mice were co-transferred 3 weeks after vaccinationinto tumor-bearing and vaccinated recipient mice (FIG. 7A). Functions ofTrp-1- and E7-specific CD8⁺T cells from spleens of wt and PPAR-α KO miceas % of cells producing 3, 2 and 1 factors right before transfer weredetermined (data not shown). Prior to transfer, functions andpolyfunctionality of Trp-1-specific CD8+T cells are similar between thetwo groups, while E7-specific CD8+T cells are less abundant andpolyfunctional in PPARα KO mice. FA catabolism of CD8+ TILS is decreasedthrough PPAR-α knockout. This may reflect that strength of TcRsignaling, which is lower for the E7 epitope, affects to what degree andat what time after activation cells rely on fatty acid catabolism.Expression of CD127 is comparable between the two T cell subsets,indicating no major differences in memory formation (histograms notshown). A heatmap comparing transcripts of TCA cycle and lipidcatabolism enzymes catabolism enzymes in CD44⁺CD8⁺TILs derived fromPPAR-α KO and wt donor mice 3 wk post transfer (n=3-4 samples/group) wasreview (data not shown) and CD44+CD8+TILs from PPARα KO as compared towt donors collected from recipient mice 3 weeks after transfer show atranscriptional profile similar to that of CD8+T cells cultured in vitroin Gal medium under hypoxia. This indicates reduced FA catabolism byPPARα KO-derived CD8+TILs. They show lower levels of PD-1 expressionconcomitant with decreases in frequencies and functions includingpolyfunctionality (FIGS. 7B, 7C). Collectively these data confirm thatFA catabolism promotes PD-1 expression but preserves CD8+T cell effectorfunctions upon metabolic stress.

Example 6: Treatment with Anti-Pd-1 Slows Tumor Progression withoutChanging CD8+ TILs' Metabolism or Functions

In clinical trials checkpoint inhibitors such as monoclonal antibodies(mAb) to PD-1 can delay tumor progression (Larkin et al., 2015). As inthe model CD8+TILs increase PD-1 expression over time, we tested iftreatment with anti-PD-1 mAb affects their metabolism or functions.

Mice were challenged with tumor cells, vaccinated 3 days later, andstarting 10 days after vaccination treated with anti-PD-1 mAb or isotypecontrol. Within 1-month tumors, anti-PD-1 treatment reduces staining forPD-1 and enhances pAkt levels on/in vaccine induced CD8+TILs (FIG. 5A)(Patsoukis et al., 2013) without affecting their differentiation status(FIG. 5B). PD-1 blockade neither dramatically affects the FA or Glucatabolism of CD44+CD8+TILs (FIGS. 5C, 5D and 5E) nor improves effectorfunctions of vaccine-induced CD8+TILs (FIG. 5F).

Graphs (not shown) of tumor growth in vaccinated C57Bl/6 mice vs.unvaccinated C57Bl/6 mice and immune-deficient NSG mice, which lack T, Band natural killer cells mice show that PD-1 blockade does not affectCD8+ TILs' functions, but it delays tumor progression althoughindependent of T cells. It does effectively delay tumor progression invaccinated as well as unvaccinated or even NSG mice, suggesting thatPD-1 checkpoint blockade delays tumor progression in a Tcell-independent manner.

Recent studies show that anti-PD-1 treatment decreases tumor progressionby reducing mTOR signaling in PD-1+ melanoma cells (Kleffel et al.,2015). As mTOR signaling increases the T cells' Glu metabolism (Palmeret al., 2015), we tested whether anti-PD-1 mAbs reduce the tumor cells'Glu metabolism and thereby delay their growth. In all three models,anti-PD-1 treatment increases Glu content within the tumors'interstitial fluid (FIG. 11A). Cells from B16BrafV600E tumors of NSGmice upon anti-PD-1 treatment increase incorporation of Glu-derivedcarbons into metabolites of the TCA cycle or the purine synthesispathway, indicating that PD-1 blockade actually increases their use ofGlu for both catabolic and anabolic reactions (FIGS. 11B, 11C).

Anti-PD-1 cause delays in tumor progression in absence of CD8+ T cells,possibly because of blockade of reverse signaling. Ligation of PD-1 toPDL1 on tumor cells increases their resistance to apoptosis or T cellmediated cytolysis. (FIG. 11D).

In summary, PD-1 blockade provides clinical benefits to cancer patientspresumably by rescuing T cell functions. In our model, conditions thatdecrease PD-1, e.g., hypoxia, decrease T cell functions; whilefenofibrate increases PD-1 expression but results in improved T cellfunction. PD-1 may be helpful in a glucose-poor environment by reducingthe T cells' reliance on glycolysis and thus preserving their functions.In the model PD-1 signaling has no major effects on metabolism orfunctions of CD8+TILs. Anti-PD-1 treatment could reduce tumorprogression in a T cell independent manner.

Combining metabolic reprogramming with PD-1 blockade is one embodimentof a useful method of treatment.

Example 7: Enhanced Reliance on FA Catabolism is Essential to MaintainFunctions of CD8+T Cells

To further assess the impact of FA catabolism on CD8+T cell functions,we stimulated CD8+T cell in presence of fenofibrate (FF), an agonist ofPPARα that increases FA catabolism, or etomoxir (Eto), an irreversibleinhibitor of Cpt1 that decreases mitochondrial FAO. In vitro FF-treatedcells stimulated in Glu or Gal medium compared to diluent-treated cellsincrease FA catabolism as shown by their enhanced transcripts of factorsinvolved in FA catabolism (PPAR-α, Ehhadh, Acox1—strongly increased forGlu, Cpt1a and Bdh1 also increased for Glu; Ehhadh and Cpt1a stronglyincreased—increased for Gal and PPAR-α, Acox1 and Bdh-1 also increased;data not shown) and increased FA uptake (FIG. 12A). Eto decreases OCR byCD8+T cells stimulated in either Glu or Gal media (FIG. 12B). OCRdeclines more in cells cultured with Gal and Eto, and further decreaseswhen cells are also subjected to hypoxia, again confirming the cells'increased reliance on FAO when Glu and O₂ are limited. Under hypoxia,PD-1 expression increases upon addition of FF but decreases in presenceof Eto (FIG. 12C). FF increases while Eto decreases functions andpolyfunctionality of CD8+T cell cultured with limited access to Glu andO₂ (FIG. 12D). These results show that enhanced FA catabolism promoteseffector functions of metabolically stressed CD8+T cells, although itincreases PD-1 expression.

To assess how increased FA catabolism affects CD8+TIL functions, wevaccinated CD90.2+ mice congenic for CD45, and treated them for 3 weeksdaily with FF (CD45.1 mice) or diluent (CD45.2 mice). Splenocytes fromthese mice were mixed at a 1:1 ratio of Trp-1-specific CD8+T cells fromthe 2 donors and transferred into CD90.1+ recipient mice, which had beenchallenged with tumor cells and vaccinated 3 days later. Cells weretransferred 2 days after vaccination (FIG. 6A). Immediately beforetransfer FF- and diluent treated Trp-1- and E7-specific CD8+T cells fromdonor mice show comparable expression of CD62L, CD127, KLRG1 and FoxO1,indicating that FF does not affect memory formation (FIG. 6B).FF-treated cells show increased expression of PD-1 and T-bet, suggestiveof a higher activation status. FF treatment does not significantlyenhance frequencies or functions of vaccine-induced CD8+T cells (FIG.6C). FF-treated splenocytes before transfer show enhanced OCR, which isblocked by Eto indicating that FF conditions vaccine-induced CD8+T cellsto enhance FAO (FIG. 6D).

Donor-derived vaccine-induced CD8+TILs were analyzed 3 weeks aftertransfer (data not shown). Compared to diluent-treated TILs of donororigin, FF-treated donor derived CD44+CD8+TILs show enhanced levels oftranscripts for factors involved in FA catabolism (data not shown). BothTrp-1- and E7-specific FF donor-derived CD8+TILs show a trend towardsincreased PD-1 expression (FIG. 6E). Frequencies and functions ofFF-treated, vaccine-induced CD8+TILs of donor origin are significantlyhigher compared to those of controls (FIG. 6F). Upon transfer ofsplenocytes from FF- or diluent-treated mice into separate cohorts oftumor-bearing mice, the former significantly delays tumor growth (FIG.6G). Collectively these data confirm that enhanced FA catabolismimproves antitumor functions of CD8+TILs.

To test if FF-induced PD-1 increases affect FF-treated CD8+TILfunctions, we fed vaccinated donor mice with FF or diluent daily forthree weeks and then upon transfer into separate tumor-bearing micetreated the recipients with anti-PD-1 or isotype control antibodies(FIG. 6H). Both FF treatment of donors and anti-PD-1 treatment ofrecipients strongly delay tumor progression (FIG. 6I). Moreover, theyact synergistically and together completely prevent tumor outgrowth inmore than 30% of the vaccinated mice (FIG. 6I and not shown). Anti-PD-1treatment reduces PD-1 staining on donor cells and this is not affectedby FF (FIG. 6J). It only has subtle effects on frequencies and functionsof MAA-specific CD8+TILs derived from either set of donor mice (data notshown). PD-1 blockade significantly increases frequencies ofmonofunctional E7-specific CD8+TILs derived from FF treated donors. Thiseffect may partially reflect the smaller tumor sizes of α-PD-1 treatedmice, which might rescue the bystander T cell functions more easily thanthose of MAA-specific CD8+TILs.

To further study how FA catabolism affects functions of CD8+TILs, westimulated CD8+T cells from PPARα KO mice in vitro and compared them tothose from wildtype (wt) mice. Transcripts for most factors involved inthe TCA cycle and lipid metabolism (e.g., IDH3a, MDH2, slc27a2, slc27a4,LIPA, Acaa1a, Acox1, Cpt1a and Acadvl) are higher in PPARα KO comparedto wt CD8+T cells when stimulated in Glu medium and under hypoxia; thisprofile reverses in cells cultured in Gal medium and low O₂, suggestingthat PPARα inactivation significantly decreases FA catabolism by CD8+Tcells cultured without Glu (data not shown). PPARα KO compared to wtCD8+T cells express lower levels of PD-1 when cultured in Gal-mediumregardless of O₂ levels (FIG. 13A). Their functions andpolyfunctionality are significantly lower compared to those of wt CD8+Tcells cultured in Gal-medium (FIG. 13B), suggesting that FA catabolismis required to maintain effector functions of CD8+T cells activated withlimited access to Glu.

We explored the effect of PPARα inactivation on vaccine-induced TILfunctions in an adoptive transfer system, in which splenocytes fromPPARα KO and wt mice were collected 3 weeks after vaccination, mixed ata 1:1 ratio of Trp-1-specific CD8+T cells from the two donors andco-transferred into tumor-bearing and vaccinated recipient mice (FIG.7A). Prior to transfer, functions and polyfunctionality of MAA-specificCD8+T cells are similar between the two groups, while E7-specific CD8+Tcells are less abundant and polyfunctional in PPARα KO mice (data notshown). This difference may reflect that strength of T cell receptor(TCR) signaling, which is lower for the E7 epitope, affects at what timeafter activation cells switch to FA catabolism. Expression of CD127 iscomparable between the two T cell subsets, indicating no majordifferences in memory formation (FIG. 7C). CD44+CD8+TILs from PPARα KOas compared to wt donors collected from recipient mice 3 weeks aftertransfer show a transcriptional profile similar to that of PPARα KOCD8+T cells cultured in vitro in Gal medium under hypoxia, indicatingreduced FA catabolism by PPARα KO CD8+TILs (figure not shown; butexpression of IDH3a, MDH2, slc27a2, slc27a4, LIPA, Acaa1a, Acox1, Cpt1a,Acadvl were decreased in the T cells from the PPAR-α KO mice compared tothe wildtype). Both Trp-1- and E7-specific PPARα KO CD8+TILs show lowerlevels of PD-1 expression, concomitant with decreases in frequencies andfunctions including polyfunctionality comparing to those ofdonor-derived wt CD8+TILs (FIG. 7B and FIG. 7C). Collectively these dataconfirm that FA catabolism preserves CD8+T cell effector functions undermetabolically challenging conditions regardless of PD-1 expressionlevels.

Example 8: Usefulness of Pretreated T Cells in Treatment of Cancer

The inventors have shown that fenofibrate treatment during in vitrostimulation of OT-1 cells causes metabolic switching of T cellmetabolism (FIGS. 15A-17E). CD8+ T cells from OT-1 mice have a TCR thatrecognizes the OVA-derived peptide SIINFEKL SEQ ID NO: 1. See, e.g.,Clarke, S R et al, 2000 Characterization of the ovalbumin-specific TCRtransgenic line OT-I: MHC elements for positive and negative selection,Immunol. Cell Biol, 78(2):110-117. Therefore OT-1 cells are a good modelof T cells that can be stimulated with any antigen, in this case theOVA-derived peptide, rather than a tumor-specific antigen.

To assess the effect of a T cell or T cell population that waspretreated in vitro with a compound or reagent that promotes the use offatty acid catabolism rather than glucose for energy production, i.e.,fenofibrate, on tumor growth in vivo, the following experiment wasconducted.

Mice were injected with 10⁵ B16.F10 melanoma cells that were transducedto express the immunodominant epitope of ovalbumin SIINFEKL SEQ IDNO: 1. Five days later the mice received either

-   -   (a) untreated OT-1 CD8+ T cells (i.v.) which express a        transgenic T cell receptor for SIINFEKL (naïve OT-1 transfer),    -   (b) OT-1 cells that were stimulated/activated in vitro with the        SIINFEKL peptide and pre-treated with diluent (Ctrl treated        OT-1), or    -   (c) OT-1 cells that were stimulated/activated in vitro with the        SIINFEKL peptide and pretreated with 25 μM of fenofibrate (FF).

Tumor growth in the mouse models was monitored for up to 24 days. Theresults are depicted in the graph of FIG. 16 showing that the growth ofthe tumor in mice treated with FF-pre-treated, activated T cells wasinhibited compared to the response in the naïve, non-activated OT-1cells or the activated, control-treated T cells.

Thus, the pretreated, activated T cell populations, such as CAR-T cells,or others, can have their metabolic functions switched and, whenadministered to a subject with a tumor, act to repress tumor growth.Such effect can be accomplished by administering pre-treated T cellsalone, i.e., without any other anti-cancer immunotherapeutic, such as atumor antigen specific vaccine. Thus the methods and compositionsdescribed herein are useful in adoptive T cell transfer therapeutictreatments using T cells, e.g., CAR-T cells, pretreated to switch theirmetabolic functions, resulting in enhanced treatment of cancer patients.

In summary, the data presented herein demonstrates that metabolismprofoundly affects T cell differentiation and effector functions. Withinthe TME CD8⁺T cells experience hypoxia and have to compete fornutrients, especially Glu, which tumor cells consume to fuel glycolysis.Cells can compensate for lack of Glu by switching from glycolysis toOXPHOS, using alternative nutrients such as FAs. Recent studies reportthat hypoglycemia within the TME impairs CD8+T cells functions andreduces the efficacy of active immunotherapy (Chang et al., 2015; Ho etal., 2015). Results presented here show that metabolic challenges withinthe TME impair the performance of CD8⁺TILs including bystander TILsalthough TA-specific CD8⁺TILs tend to be more affected. This reflectsthat TA-specific CD8⁺TILs continue to receive stimulatory signals withinthe TME as evidenced by their proliferation. In addition, they maypenetrate more deeply into tumors where nutrients and O₂ are especiallylimiting.

Solid tumors develop areas of hypoxia, which activates the HIF-1αpathway in cells of the TME. HIF-1α expression also rises upon T cellactivation²⁰. In the study HIF-1α increases in both MAA-specific andbystander CD8⁺TILs, pointing towards hypoxia as the underlying cause.The effect of hypoxia on CD8⁺T cells is controversial. Some studies showthat O₂ is required for T cell effector functions^(28, 30). Others usingprotocols in which CD8⁺ T cells were subjected to hypoxia during aresting period report that hypoxia increases functions^(10,11). the dataagree with the former as they show reduced HIF-1α signaling improvesCD8+TIL frequencies and functions, indicating that when Glu is limiting,promoting glycolysis and inhibiting OXPHOS by HIF-1α becomes detrimentalto CD8+TILs. LAG-3, which according to thethe data is regulated byHIF-1α inhibits T cell expansion and effector functions (Grosso et al.,2007). The LAG-3 locus has several HIF-1α response elements ([A/G]CGTA,(Pescador et al., 2005), which may influence LAG-3 expression underhypoxia. we show that hypoxia dampens effector functions of activatedCD8⁺T cells in vitro, and reduced HIF-1α signaling in CD8⁺TILs improvestheir frequencies and functions. The latter most likely reflects thatwhen Glu is limiting within the TME, promotion of glycolysis andinhibition of OXPHOS by HIF-1α becomes detrimental. LAG-3, whichaccording to the data is regulated by HIF-1α inhibits T cell expansionand effector functions¹². An analysis of sequences of the LAG-3 locusreveals several HIF-1α response elements ([A/G]CGTA)²⁷, which mayinfluence LAG-3 expression.

Hypoxia and hypoglycemia send opposing metabolic signals. The formerpromotes glycolysis while the latter forces cells to use OXPHOS, whichcan be fueled by various nutrients but requires O₂. the Cancer cellsincrease de novo lipogenesis²¹ and recruit adipose progenitor cells³⁹.Accordingly in thethe model the abundance of free FA species increasesduring tumor progression. thethe data show that CD8⁺TILs cope with lackof Glu and O2 by augmenting FA uptake and FA catabolism to gain energythrough OXPHOS. However, even with this metabolic switch CD8+TILs showloss of functions, which can be improved by further promoting lipidmetabolism by FF.

High expression of PD-1 is viewed to signal CD8⁺T cell exhaustion andloss of effector functions. The results suggest that high PD-1expression is not inevitably linked to impaired T cell functions. Whenactivated CD8⁺T cells are exposed to hypoxia, their decreased expressionof PD-1 is associated with impaired functions. In contrast, FF-treatedCD8⁺T cells show a trend towards increased PD-1 expression but theirfunctions improve PD-1 signaling inhibits TCR- and CD28-mediatedactivation of the PI3K/Akt/mTOR pathway, which in turn decreasesglycolysis (Parry et al., 2005) and promotes lipolysis and FAO(Patsoukis et al., 2015). We speculate that enhanced PD-1 signaling inCD8+TILs is beneficial by facilitating their metabolic switch within aGlu-poor TME. In the model blockade of PD-1 after the initial phase of Tcell activation affects neither functions nor metabolism of TILsalthough overall Glu concentrations within the tumors increase. Theseresults differ from those of a recent study in a mouse sarcoma model,which reports improved glycolysis and IFN-γ production by CD8+TILstreated with anti-PD-1 during their initial activation (Chang et al.,2015). These apparently opposing results reflect intrinsic differencesin tumor models or in T cells induced by vaccination or throughstimulation by tumor-derived antigens. Alternatively, differences intiming of treatment may affect the result. PD-1 blockade during theinitial stages of T cell activation may allow them to better compete forGlu within a TME; once TILs have switched to FA catabolism they remaincommitted to this pathway regardless of PD-1 signaling.

Anti-PD-1 treatment delays tumor progression in the model. Some of thedata suggest that anti-PD-1 may promote MAA-specific CD8+T cellinfiltration into tumors (not shown). However, as anti-PD-1 treatmentalso delays tumor progression in immune-deficient mice, we assume thatit acts directly on tumor cells, tumor stromal cells orimmunosuppressive cells within the TME. A recent study suggests thatanti-PD-1 may reduce proliferation of PD-1+ tumor cells by blocking mTORsignaling (Kleffel et al., 2015). This mode of action of PD-1 blockadewill only affect PD-1+ tumor cells. As the melanoma cells isolated fromtumors grown in vivo express very low levels of PD-1 (data not shown);we view it as unlikely that they are directly affected by PD-1 blockade.Immunosuppressive cells express high levels of PD-1 (not shown) and PD-1blockade may impair their ability to promote tumorigenesis (Marvel andGabrilovich, 2015). Melanoma cells express PD-L1 (not shown) andback-signaling through this ligand increases the tumor cells' resistanceto Fas- or CD8+T cell mediated apoptosis (Azuma et al., 2008). Anti-PD-1treatment could thus promote tumor cell death by enhancing theirsusceptibility to apoptosis or, in immunocompetent mice, indirectlyimprove TIL functions by increasing the tumor cells' susceptibility tolytic enzymes. Either mechanism could delay tumor growth and thusenhance levels of Glu within the TME. Although the additional Glu couldfuel proliferation of tumor cells this would be counterbalanced by theirincreased death rates.

Energy production through FAO rather than glycolysis comes at a price;more O₂ is needed to generate equivalent amounts of ATP and ROSproduction increases. Generating energy through FAO within a hypoxic TMEmay thus not be the only method by which CD8+TILs maintain theirfunctions. Ketone bodies are highly efficient fuels that require less O₂(Veech, 2004) and previous studies showed that they serve as thepreferred energy source for cells of the nervous system subjected tohypoxia and hypoglycemia (Takahashi et al., 2014).

Ketone bodies could be synthesized and secreted by other cells(Martinez-Outschoorn et al., 2012), or they could be produced by TILsdirectly as suggested by increased transcript levels of Bdh1, a keyenzyme in ketone body metabolism. The data show that CD8+TILs showpronounced increases in the intensities of ketone bodies acetoacetateand 3-hydroxybutyrate during tumor progression. Additionally, levels ofO₂ differ within a tumor and TILs can randomly migrate within the TME(Mrass et al., 2006). TILs could use FAO and ketone bodies alternativelydepending on surrounding O₂ levels to maintain their effector functionsand prolong their survival.

As suggested by the results, metabolic reprogramming of CD8+T cells toincrease energy production through FA catabolism prior to adoptive celltransfer enhances the overall efficacy of cell therapy in patients withsome types of cancers, especially those characterized by low Glu contentlike melanomas. In agreement, other studies show that memory CD8+Tcells, which prefer FAO and OXPHOS for energy production, are better atslowing tumor progression than effector cells (Crompton et al., 2015;Sukumar et al., 2013). In contrast, others report that increasing theTILs' ability to use glycolysis improves their antitumor effect (Changet al., 2015). Which metabolic manipulations are most suited to improveTIL-mediated tumor regression will likely depend on the nature of thetumor. Those with sufficient levels of Glu may benefit from CD8+T cellswith high glycolytic potential, while tumors with a hypoglycemic TME maybest be combated by CD8+T cells that favor FA catabolism.

The results herein show that metabolic challenges within the TME haveprofound impacts on CD8+TILs. It forces CD8+TILs to increasingly gainenergy through FA catabolism, including consumption of ketone bodies,which partially preserves their functions and may improve theirsurvival. Promoting the CD8+ TILs' propensity to use FAO combined withPD-1 signaling blockade further improves treatment outcome. Theseresults invite further investigations to assess if the outcome of cancerimmunotherapy can be improved by adding metabolic manipulations tocurrent treatment strategies.

Data suggest that high PD-1 expression is not inevitably linked toimpaired T cell functions. PD-1 inhibits T cell receptor- andCD28-mediated activation of the mTOR pathway, which in turn decreasesglycolysis²⁴ and promotes lipolysis and fatty acid catabolism²⁵. PD-1may be regulated by environmental cues, which direct CD8⁺T cells toadjust their metabolism. Under hypoxia when Glu is available, glycolysisprovides an easy source of O₂-independent energy, which requires reducedPD-1 expression. When Glu and O₂ are limiting, CD8⁺T cells increase PD-1expression, which facilitates FA metabolism.

As supported by the results, metabolic reprogramming of CD8⁺T cells toincrease energy production through FA catabolism prior to adoptive celltransfer enhances the overall efficacy of cell therapy in patients withsome types of cancers, especially those characterized by low Glucontent, like melanomas. In agreement, other studies show that memoryCD8⁺T cells, which prefer fatty acid catabolism and OXPHOS for energyproduction, are better at slowing tumor progression than effectorcells³¹′⁸. In contrast, others report that increasing the TILs' abilityto use glycolysis improves their antitumor effect⁶. Which metabolicmanipulations are most suited to improve TIL-mediated tumor regressiondepends on the nature of the tumor. Those with sufficient levels of Glumay benefit from CD8⁺T cells with high glycolytic potential while tumorswith a hypoglycemic TME may best be combated by CD8⁺T cells, which favorFA catabolism.

Thus, the inventors have shown that metabolic stress within the TME hasprofound effects on CD8⁺TILs. It forces CD8⁺TILs to increasingly gainenergy through FA catabolism, including consumption of ketone bodies,which preserves their functions and may improve their survival. Thusenhancing the FA metabolism of tumor infiltrating CD8+ T cells increasesthe efficacy of active immunotherapy of cancers such as melanoma. Theoutcome of immunotherapy can be improved by metabolic manipulations.

The inventors have determined that treatment with an anti-PD-1 mAb slowstumor progression without changing CD8+ TILs' metabolism or functions.Treatment with anti-PD-1 effectively delays tumor progression invaccinated as well as unvaccinated or even immune-deficient NSG mice,which lack T, B and natural killer cells, suggesting that PD-1checkpoint blockade delays tumor progression in a T cell-independentmanner.

Anti-PD-1 treatment acts synergistically with metabolic reprogramming ofTILs to achieve superior antitumor efficacy. Both fenofibrate treatment(FF—an agonist of PPAR-alpha that increases FA catabolism) of donors andanti-PD-1 treatment of recipients strongly delay tumor progression andact synergistically together to completely prevent tumor outgrowth inmore than 30% of vaccinated mice (FIGS. 6I-6J).

FA catabolism improves T cell functions through multiple mechanisms: Itcontributes to energy production. It contributes to amino acid synthesisand production of effector molecules. It contributes to acetyl-CoA poolin the cytosol, acetylates GAPDH and enhances IFN-γ translation. Thedata demonstrates that metabolic manipulations that conditiontumor-associated-specific T cells to optimally cope with the metabolicconstraints within the TME can significantly improve the overallefficacy of cancer therapy.

TABLE 3 (Sequence Listing Free Text) The following information isprovided for sequences containing free text under numeric identifier<223>. SEQ ID NO: (containing free text) Free text under <223> 2 Forwardprimer 3 Reverse primer 4 Forward primer 5 Reverse primer 6 Forwardprimer 7 Reverse primer 8 Forward primer 9 Reverse primer 10 Forwardprimer 11 Reverse primer 12 Forward primer 13 Reverse primer 14 Forwardprimer 15 Reverse primer 16 Forward primer 17 Reverse primer 18 Forwardprimer 19 Reverse primer 20 Forward primer 21 Reverse primer 22 Forwardprimer 23 Reverse primer 24 Forward primer 25 Reverse primer 26 Forwardprimer 27 Reverse primer 28 Forward primer 29 Reverse primer 30 Forwardprimer 31 Reverse primer 32 Forward primer 33 Reverse primer 34 Forwardprimer 35 Reverse primer 36 Forward primer 37 Reverse primer 38 Forwardprimer 39 Reverse primer 40 Forward primer 41 Reverse primer 42 Forwardprimer 43 Reverse primer 44 Forward primer 45 Reverse primer 46 Forwardprimer 47 Reverse primer

Each and every patent, patent application, including priority U.S.provisional application Nos. 62/279,252, 62/419,775, and 62/420,271,filed Jan. 15, 2016, Nov. 9, 2016 and Nov. 10, 2016, respectively, andeach and every publication, including websites cited throughout thedisclosure and listed herein, and the Sequence Listing accompanying thisapplication, is expressly incorporated herein by reference in itsentirety. While this invention has been disclosed with reference tospecific embodiments, it is apparent that other embodiments andvariations of this invention are devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims include such embodiments and equivalent variations.

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1. A method for treating cancer comprising administering to a subjecthaving a cancer a T cell or T cell population that is pretreated orconditioned ex vivo or in vitro with a compound or reagent that promotesthe use of fatty acid catabolism rather than glucose for energyproduction by the pre-treated T cells.
 2. The method according to claim1, further comprising co-administering to a subject having a cancer: (a)an immunotherapeutic composition targeting an antigen or ligand on atumor cell in the subject; and (b) one or more of i. a compound orreagent that promotes the use of fatty acid catabolism by tumorantigen-specific T cells in the tumor microenvironment; and ii. a T cellpretreated ex vivo with (i) to condition the T cell to use fatty acidsrather than glucose for energy production for adoptive cell transfer. 3.A method of modifying a T cell comprising pretreating the T cell ex vivoor in vitro with a compound or reagent that conditions the cell to usefatty acid catabolism for energy production by the T cells.
 4. Themethod according to claim 3, for enhancing the survival of a chimericantigen receptor-T cell or a chimeric endocrine receptor-T cell or an exvivo expanded tumor antigen-specific T cells further comprisingpretreating the T cell ex vivo with a compound or reagent that promotesthe use of fatty acid catabolism for energy production by tumorantigen-specific T cells in the tumor microenvironment before adoptivecell transfer to a subject having a solid tumor.
 5. The method accordingto claim 2, which is a therapeutic regimen for the treatment of cancercomprising: (a) administering to a subject having a cancer characterizedby a solid tumor a single dose of an immunotherapeutic compositiontargeting an antigen or ligand on the tumor cell on a day 1 oftreatment; (b) administering to said subject a compound or reagent thatpromotes the use of fatty acid catabolism by tumor antigen-specific Tcells in the tumor microenvironment, said first dose of the fatty acidcatabolism-promoting compound of reagent beginning on day 0-5 oftreatment; (c) administering the fatty acid catabolism-promotingcompound or reagent daily from the beginning day of treatment of (b)until a day occurring between day 7 to day 30 of treatment.
 6. Themethod according to claim 1, further comprising administering acheckpoint inhibitor in the form of an antibody or a small molecule. 7.The method according to claim 6, wherein the checkpoint inhibitor is ananti-PD-1 antibody or small molecule ligand.
 8. The method according toclaim 1, wherein the compound or reagent that promotes the use of fattyacid catabolism by T cells is fenofibrate, clofibrate, gemfibrozil,ciprofibrate, bezafibrate, an AMPK activator or5-aminoimidazole-4-carboxamide riboside.
 9. The method according toclaim 1, wherein the T cell is an autologous or heterologous, naturallyoccurring T cell or a recombinantly or synthetically modified T cellconstruct, or a human T cell or natural killer (NK) T cell or Tinfiltrating lymphocyte (TIL) obtained from the subject or from a bonemarrow transplant match for the subject, or a T cell obtained from humanperipheral blood or from the tumor microenvironment of the subject, or aT cell modified to express a heterologous antigen receptor, or achimeric antigen receptor or a chimeric endocrine receptor prior to saidpretreatment, or an endogenous or heterologous human T cell or human Tcell line, or a CD8+ T cell.
 10. The method according to claim 2,wherein said immunotherapeutic composition is a recombinant virus orvirus-like particle that expresses a cancer antigen, a DNA constructthat expresses a cancer antigen, a composition comprising cancerantigens or fragments thereof as peptides or proteins, monoclonalantibodies or antigen-binding fragments that specifically bind cancerantigens.
 11. The method according to claim 1, wherein: (a) theimmunotherapeutic composition and the fatty acid catabolism-promotingcompound or reagent, or the immunotherapeutic composition and thepretreated T cell are administered substantially simultaneously; or (b)the fatty acid catabolism-promoting compound or reagent or thepretreated T cells are administered once or repeatedly from at least oneto 14 days after administration of the immunotherapeutic composition; or(c) the immunotherapeutic composition is administered in a single doseor as one or more booster doses; or (d) said composition (a) and (b)(i)are independently administered systemically by intramuscular,intraperitoneal, intravenous, intratumoral or intranodal administration;or (e) composition (b) is administered orally.
 12. The method accordingto claim 1, wherein the pretreated T cells are administered once orrepeatedly or wherein the pretreated T cells are administered in asingle dose or as one or more doses or wherein the pretreated T cellsare administered systemically by intravenous injection or infusion. 13.The method according to claim 1, further comprising (a) treating thesubject with other anti-cancer therapies; and (b) treating the subjectwith chemotherapy before administering the pre-treated T cells.
 14. Themethod according to claim 13, wherein the anti-cancer therapy comprisesdepleting the subject of lymphocytes and optionally surgically resectingthe tumor prior to administration of the pretreated T cells.
 15. Themethod according to claim 1, wherein the cancer or tumor targeted by themethod is characterized by hypoxia, significant infiltration with Tlymphocytes, and low glucose in the tumor microenvironment.
 16. Acomposition comprising a T cell that has been pretreated ex vivo or invitro with a compound or reagent that conditions the cell to use fattyacid catabolism for energy production by the T cells.
 17. Thecomposition according to claim 16, wherein (a) said composition is foruse in a method of adoptive transfer to a mammalian subject; (b) thecompound or reagent that conditions the cell to use of fatty acidcatabolism is fenofibrate, clofibrate, gemfibrozil, ciprofibrate,bezafibrate, an AMPK activator or 5-aminoimidazole-4-carboxamideriboside; or (c) the T cell is an autologous or heterologous, naturallyoccurring T cell or a recombinantly or synthetically modified T cellconstruct, or a human T cell or natural killer (NK) T cell or Tinfiltrating lymphocyte (TIL) obtained from the subject or from a bonemarrow transplant match for the subject, or a T cell obtained from humanperipheral blood or from the tumor microenvironment of the subject, or aT cell modified to express a heterologous antigen receptor, or achimeric antigen receptor or a chimeric endocrine receptor prior to saidpretreatment, or an endogenous or heterologous human T cell or human Tcell line, or a CD8+ T cell.